Biocomposite materials and related compositions, methods and systems

ABSTRACT

Biocomposites and related fabrication methods and systems are described, the biocomposites comprising compacted plants and/or algae cells having a water content of less than 15 wt %, and a minimized pore presence and/or dimensions, in which the compacted cells are in a lamellar stacked configuration with a plurality of lamellae arranged one above the other, each lamella independently having a thickness of 20 nm to 5 μm and comprising a semi-crystalline structure formed by biopolymers of cell walls of the compacted plant and/or algae cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/865,506 entitled “Bio-Composite Materials From Plant Cells,Algae, And Agriculture Waste” filed Jun. 24, 2019, which is incorporatedherein by reference in its entirety.

FIELD

The present disclosure relates to a biocomposite material and related,compositions, devices, methods and systems. In particular, the presentdisclosure relates to materials combinations and methods to produce anovel class of biocomposites from a sustainable source including plantcells, algae, and agricultural waste.

BACKGROUND

The emerging need for materials that are sustainable from anenvironmental point of view has led to the introduction of biologicalpolymers and fibers in composites to limit the amount of non-degradablecomponents and reduce their impact on the environment.

Despite the most recent efforts to in providing on new sustainablematerials has focused on utilizing fibers and fillers from sustainableand renewable resources as reinforcements, introducing recycled andwaste coproducts, and developing materials with desirable physical andmechanical properties from an environmentally sustainable source remainschallenging.

SUMMARY

Described herein is a fabrication method and related biocompositescompositions, methods, and systems comprising cultured plant and/oralgae cells that in several embodiments provide environmentallysustainable materials further possessing unexpectedly mechanical andother physical properties comparable or superior to existingbiocomposite and fossil based materials.

According to a first aspect, a biocomposite is described comprisingcompacted plant and/or algae cells in a lamellar stacked configurationwith a plurality of lamellae arranged one above the other, each lamellaindependently having a thickness of 20 nm to 5 μm.

In each lamella of the biocomposite of the disclosure compacted cellswalls of the plant and/or algae cells form a semicrystalline structurehaving a crystalline component of glucose-based biopolymers within anamorphous matrix of sugar-based biopolymers and/or phenol-basedbiopolymers of cell walls of the compacted plant and/or algae cells. Inthe biocomposite, the compacted plant and/or algae cells have a watercontent of less than 15 wt %. In the biocomposite, the compacted plantand/or algae cells are poreless or have pores with a diameter of lessthan 10 μm.

According to a second aspect, a biocomposite fabrication method isdescribed for providing a biocomposite according to the presentdisclosure and a biocomposite obtained thereby.

The method comprises: compacting along a plane a cultured biomasscomprising cultured plant and/or algae cells from a suspension culture,the cultured plant and/or algae cells having a water content of at least10 wt %, and a turgor pressure,

In the method the compacting is performed by continuously applying tothe cultured biomass an increasing pressure perpendicular to the planeuntil reaching an applied pressure corresponding to the turgor pressureof the cultured plant and/or algae cells to provide a compressedbiomass.

In the fabrication method the compacting further comprises drying thecompressed biomass by one or both of

-   -   a) applying a drying pressure to the compacted biomass and/or    -   b) applying a drying temperature to the compressed biomass to        obtain a biocomposite material having water content of 0.1-15 wt        %, a mass loss of 80-99% and/or a dry density of 500-1500 kg/m³.

According to a third aspect, a biocomposite fabrication system isdescribed, comprising a cultured biomass comprising cultured plantand/or algae cells from a suspension culture having a water content ofat least 10 wt %, and a compression tool.

In the system, the biomass comprises cultured plant and/or algae cellsand the compression tool is configured to interact with the culturedbiomass in accordance with the fabrication method herein described toprovide a biocomposite material having water content of 0.1-15 wt %, amass loss from the cultured biomass of 80-99 wt % and/or a dry densityof 500-1500 kg/m³.

The biocomposites and related materials, compositions, methods andsystems herein described, allows in several embodiments to providematerials from sustainable and renewable resources such as recycled andwaste bioproducts, and having a flexural modulus ranging from 0.1 to 7GPa, modulus of rupture 5-95 MPa, Young's modulus 1.5-5 GPa, tensilestrength ranging from 9 to 35 MPa, and/or compressive modulus 0.04-1.5GPa.

The biocomposites and related materials, compositions, methods andsystems herein described, allows in several embodiments to producebiocomposite from renewable (naturally replenished with time) and/orsustainable (which can be maintained for a definable period of time)biological resources with mechanical properties similar or superior toexisting material from the biological sources.

The biocomposites and related materials, compositions, methods andsystems herein described, allow in several embodiments to providebiocomposites with a controllable longevity of material to be optionallyenhanced by such as application of a polymer coating to a biocompositematerial herein described.

The biocomposites and related materials, compositions, methods andsystems herein described, allows in several embodiments to providebiocomposites that can be used as construction materials with a positiveenvironmental impact by generating large quantities of the material,from renewable sources such as recycled and waste bioproducts.

The biocomposites and related materials, compositions, methods andsystems herein described, allows in several embodiments to providebiocomposites without need for treatment with a chemical (e.g.solvent/acid/base) treatment and/or addition of adhesives.

The biocomposites and related materials, compositions, methods andsystems herein described, can be used in connection with anyapplications wherein an environmentally sustainable material havingmechanical properties comparable or superior to existing fossil-basedmaterial is desired. Exemplary applications comprise materials forconstruction and/or insulation, (such as panels and bricks), as well asvarious types of packaging material including single use packagingmaterial (e.g. food containers, make up containers), multi-use packagingmaterial, such as plastic and wood alternative (e.g. plastic cafeteriatrays, furniture, clothes hangers), small scale packaging (e.g. smallvolume, small production batches), packaging material not requiringwaterproof properties, waterproof material materials, and additionalidentifiable by a skilled person. Additional uses and applicationapplications are also identifiable by a skilled person.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the description of exampleembodiments, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows schematic of an exemplary fabrication method according tothe disclosure. Plant and/or algae cells (Panel A) are cultured (PanelB), harvested (Panel C) and subjected to a controlled compression (PanelD) and dehydration, resulting in the plant cell walls (Panel E)providing a lamellar architecture when dried (Panel F).

FIG. 2 shows panel (A) photograph of the cell culture. Microscopy imagesof the cells stained for panel (B) pectins, panel (C) cellulose, andpanel (D) lignols.

FIG. 3 shows Raman spectrum of plant cells; peaks assigned to pectin(P), cellulose (C), hemicellulose (H) and monolignols (M).

FIG. 4 shows SEM cross-sectional views of the microstructure of anexemplary biocomposite of the present disclosure (Panel A) compared withmicrostructure of walnut (Panel B), MDF (Panel C) and plywood (Panel D)at the same magnification.

FIG. 5 shows SEM cross-section views of an exemplary bio-composite(Panel A), walnut panel (B) and PP panel (C) at the same magnification,showing the different microstructures of these materials.

FIG. 6 shows panel (A) photograph of an exemplary biocomposite sample,panel (B) SEM image of a cross-section, demonstrating the lamellarmicro-structure, panel (C) TEM and panel (D) HRTEM images of thebiocomposite cross-sections.

FIG. 7 shows panel (A) TEM of a cross-sectional area of an exemplarybiocomposite according to the present disclosure. Gray arrows indicatethe testing direction for tensile experiments, panel (B) selectedsubsection for tomography imaging, panel (C) 3D reconstruction ofselected cell wall subsection. Gold corresponds to dark pixels in theTEM, showing how the cell wall material is distributed in the selectedarea.

FIG. 8 shows TGA (thinner solid line) and DTG (numerically annotatedthicker dotted line) plots of the dehydrated biocomposite.

FIG. 9 shows XRD pattern with marked contributions from cellulosepolymorphs Iα, Iβ, II and III.

FIG. 10 shows density of the biocomposite and reference materials.Samples notation: BC: pure (without fillers) bio-composite; 1: pine; 2:poplar; 3: oak; 4: walnut; 5: plywood; 6: MDF; 7: PS; 8: PP; 9: LDPE.

FIG. 11 shows biodegradation of the biocomposite (BC, in square) andnatural pine (in circle).

FIG. 12 shows Young's modulus of the biocomposite and referencematerials. Samples notation: BC: pure (without fillers) bio-composite;1: pine; 2: poplar; 3: oak; 4: walnut; 5: plywood; 6: MDF; 7: PS; 8: PP;9: LDPE.

FIG. 13 shows tensile strength of the biocomposite and referencematerials. Samples notation: BC: pure (without fillers) bio-composite;1: pine; 2: poplar; 3: oak; 4: walnut; 5: plywood; 6: MDF; 7: PS; 8: PP;9: LDPE.

FIG. 14 shows flexural modulus calculated from 3-point bendingexperiments. Samples notation: BC: pure biocomposite material; 1: pine;2: poplar; 3: oak; 4: walnut; 5: plywood; 6: MDF; 7: PS; 8: PP; 9: LDPE.

FIG. 15 shows modulus of rupture calculated from 3-point bendingexperiments. Samples notation: BC: pure biocomposites material; 1: pine;2: poplar; 3: oak; 4: walnut; 5: plywood; 6: MDF; 7: PS; 8: PP; 9: LDPE.

FIG. 16 shows a comparison of mechanical properties of this work(encircled points above annotation ‘this work’), and literature-reportedbiocomposites from living, eukaryotic organisms (9, 10, 15, 31, 34, 35).

FIG. 17 shows materials properties for a biocomposite (labeled AW)produced from Nicotiana tobacco BY-2 cells: panel (A) materials density,panel (B) Young's modulus, and panel (C) tensile strength. Samplesnotation: 1: pine; 2: poplar; 3: oak; 4: walnut; 5: polystyrene; 6:polypropylene; 7: polyethylene.

FIG. 18 shows stress-strain curves obtained from exemplary biocompositesamples in tension experiments, each curve corresponding to a sample.

FIG. 19 shows stress-strain curves obtained from exemplary biocompositesamples in 3-point bending experiments, each curve corresponding to asample.

FIG. 20 shows that the biocomposites samples were subjected to 3-pointbending tests in two perpendicular directions: panel (A) in plane withthe cells lamellar arrangement and panel (B) normal to the cellsstacking plane.

FIG. 21 shows compressive modulus and strength of biocomposites with CF.Inset: SEM image of the biocomposite with 1 wt % CF (grey scale) inwhich a cross section of a CF is visible in the center of the inset.

FIG. 22 shows Young's modulus versus density for various materials andbiocomposites described herein. Upper annotation NC and upper annotationBC groups correspond to bending experiments, lower annotation BC, G,lower annotation NC and CF groups to compression. The cellulose* areacorresponds to pure cellulose fibers, papers and nanocellulose-basedproducts, including bacterial cellulose (26, 39, 40).

FIG. 23 shows IV curves for biocomposites with 1 wt % and 20 wt % CF.

FIG. 24 shows biocomposite with IN exhibiting magnetic properties.

FIG. 25 shows comparison of the Modulus of Rupture in panel (B) andFlexural Modulus in panel (A) for a biocomposite produced frommicroalgae (Chlamydomonas reinhartii), labelled “green algae”, obtainedin the presented fabrication method and the same quantities measured incommercially available particle boards.

FIG. 26 shows a schematic representation of an exemplary biocompositefabrication system of the instant disclosure.

DETAILED DESCRIPTION

Described herein is a biocomposite and the related compositions,methods, and systems based on cultured plant cells.

The term “composite” or “composite material” as used herein indicates amaterial made from two or more constituent materials with significantlydifferent physical or chemical properties that, when combined, produce amaterial with characteristics different from the individual components.In a composite material, the individual components remain separate anddistinct within the finished structure, differentiating composites frommixtures and solid solutions. Exemplary composite materials with apolymer main component (also called matrix) are found in the 2 providedreferences, mixed with different types of filler additives. (A. C.Balazs, T. Emrick and T. P. Russell, 2006; Kinloch et al., 2018). Theycombine high specific mechanical properties at low densities withprocessability, making them essential in widespread applications, fromautomotive to sports goods and robotics.

The term “biocomposite” as used herein indicates a composite material inwhich the two or more constituent materials with significantly differentphysical or chemical properties of biological origin (herein alsobiomaterials). Typically the two materials comprise a rigid biomaterialin an matrix. The rigid biomaterial can be of various biological originsand in particular can be derived from plants, such as fibers from crops(e.g. cotton, flax or hemp), recycled wood, waste paper, crop processingbyproducts or regenerated cellulose fiber (e.g. viscose/rayon). Inbiocomposites, the rigid biomaterial and its configuration are typicallymainly responsible for the mechanical properties of the biocompositewhile the matrix typically protects the rigid biomaterial fromenvironmental degradation and mechanical damage, and holds the rigidbiomaterial together and to transfer the loads on it.

Exemplary biocomposite in the sense of the disclosure can be formedliving matter, at an organism or cellular level, to serve as the maincomponent, or assemble the main component such as the biocompositereviewed and classified as Engineering Living Materials, ELM (Nguyen etal., 2018). Within ELM, composites created from living organisms andsynthetic nanomaterials, can give rise to another sub-category,nanobionics or bionicomposites, described e.g. in Pugno and Valentini,2019. L. Valentini et al., 2016; Luca Valentini et al., 2016, DiGiacomo, Maresca, Angelillo, et al., 2013; Di Giacomo, Maresca, Porta,et al., 2013; Di Giacomo, Daraio and Maresca, 2015 Haneef et al., 2017.Living organism that can form biocomposite includes for example bacteriaand more complex living organisms, such as fungi, algae and fermentingyeast and eukaryotic biological growth for production possibly as partof “self-growing” composites. The option of adding syntheticnanoparticles in the growth medium allows added properties in the finalcomposite, such as catalytic, electronic and sensing (Bigall et al.,2008) or to create hybrid panel composites of wood, fungal mycelium, andcellulose nanofibrils (CNF) with improved mechanical properties,compared to all-mycelium materials (Sun et al., 2019).

Biocomposites according to the present disclosure comprise plants and/oralgae cell, which are compacted to form a biocomposite comprising rigidbiopolymers and an amorphous matrix of biopolymers from the plant and/oralgae cell wall. The term “cell” as used herein indicates the basicstructural, functional, and biological unit of all known organisms.Cells are of two types: eukaryotic, which contain a nucleus, andprokaryotic, which do not. Most cells are only visible under amicroscope, with dimensions between 1 and 150 micrometers. Cells consistof cytoplasm enclosed within a membrane, which contains manybiomolecules such as proteins and nucleic acids.

The cell membrane is typically formed by a lipid bilayer or monolayer,including cholesterols (a lipid component) that sit betweenphospholipids to maintain their fluidity at various temperatures. Thecell membrane typically also contains membrane proteins, includingintegral proteins that go across the membrane serving as membranetransporters, and peripheral proteins that loosely attach to the outer(peripheral) side of the cell membrane, acting as enzymes shaping thecell.

The term “cytoplasm” or “cytoplasmic material” as used herein indicatesall of the material within a cell, enclosed by the cell membrane,including the nucleoplasm (cellular material inside the nucleus andcontained within the nuclear membrane). The main components of thecytoplasm are cytosol, a gel-like substance, the organelles—the cell'sinternal sub-structures, and various cytoplasmic inclusions. Thecytoplasm is about 80% water and usually colorless.

Cells in the sense of the disclosure typically comprise organelles. An“organelle” in the sense of the disclosure indicates a specializedsubunit, usually within a cell, that has a specific function. Organellesare either separately enclosed within their own lipid bilayers (alsocalled membrane-bound organelles) or are spatially distinct functionalunits without a surrounding lipid bilayer (non-membrane boundorganelles).

The term “plant and/or algae cell” as used herein indicates a eukaryoticcell comprising at least a primary cell wall and possibly a secondarycell wall, such as eukaryotic cells of a eukaryote organism of thekingdom Plantae and eukaryotic cells of any eukaryote organism of thetaxon Algae.

Plant and/or algae cells' distinctive features in the sense of thedisclosure comprises presence of i) a primary cell walls, ii) a cellmembrane, iii) internal organelles comprised within iv) cytoplasmicmaterial. Internal organelles of plant cells in the sense of thedisclosure comprise, the nucleus, a plastids, with the capability toperform photosynthesis, a vacuole that regulates turgor pressuremitochondrion, golgi apparatus and additional organelles identifiable bya skilled person.

A “cell wall” in the sense of the disclosure indicates a structurallayer surrounding some types of cells, just outside the cell membrane. Acell wall provides the cell with both structural support and protection,and also acts as a filtering mechanism A cell wall has mechanicalproperties depending on the related composition and configuration. Acell wall major function is to act as pressure vessels, preventingover-expansion of the cell when water enters.

In a “plant and/or algae cell” according to the instant disclosure, acell wall of the plant and/or algae cell is a composite which comprisesprimarily a combination of glucose based biopolymers such aspolysaccharides and glycoproteins, which provide a rigid component ofthe cell wall as well as sugar-based biopolymers and/or phenol basedbiopolymers such as. hemicellulose and/or pectin and/or a combination ofalginate carrageenan, starch and/or agar which provide the matrixcomponent of the cell wall.

As used herein, a “sugar-based biopolymer” refers to a natural polymerthat can be produced by a living organism or cell and that contains atleast three monosaccharide monomeric moieties. Exemplary monosaccharidemonomers include D-glucose units, D-mannopyranuronose,L-gulopyranuronose, and L-gulopyranuronic acid. Sugar-based biopolymerin which the at least three monosaccharide monomeric moieties areD-glucose units are indicated identified as “glucose based.” Exemplarybiopolymer of D-glucose units include cellulose. Exemplary biopolymer ofD-mannuronic acid, and L-guluronic acid includes alginic acidAdditional, exemplary sugar-based biopolymer in cell wall of a plantand/or algae cell comprises glycoproteins, polysaccharides, starches anadditional biopolymers identifiable by a skilled person.

As used herein, a phenol-based biopolymer refers to a natural polymerthat can be produced by a living organism or cell and that contains atleast three phenolic or substituted phenolic monomeric moieties.Exemplary phenolic or substituted phenolic monomers include paracoumarylalcohol, coniferyl alcohol and sinapyl alcohol.

Plant cell walls are naturally secreted by the protoplast on the outersurface of the plasma membrane of the plant cells. In particular, in aplant cell according to the disclosure the composition of cell wallsvaries between species and may depend on cell type and developmentalstage.

The primary cell wall of photosynthetic eukaryotes of the kingdomPlantae is structured as a composite typically composed of at least thepolysaccharides cellulose, hemicelluloses and pectin, in which thecellulose provides a crystalline component and the hemicelluloses andpectin are comprised in an amorphous matrix. In particular, primary cellwall of photosynthetic eukaryotes of the kingdom Plantae primarilyconsists of four building blocks, cellulose, hemicellulose, pectin andpossibly lignin in Plantae, at varying concentrations and spatialorganization depending on the plant species, the cell's stage ofdevelopment and function they serve. In those plant cells Cellulose isthe main load bearing component of the cell wall of photosyntheticeukaryotes of the kingdom Plantae and is organized into semi-crystallinemicrofibrils with excellent mechanical properties, with Young's modulusup to 220 GPa, and tensile strength up to 7.7 GPa, depending oncrystallinity and physical characteristics (16). In the cell wall ofphotosynthetic eukaryotes of the kingdom Plantae, cellulose microfibrilsare immersed in a matrix of amorphous polysaccharides, hemicellulosesand pectins (30). Hemicelluloses bind individual cellulose fibrilstogether, assisting in the load transfer within the cell wall. Pectinsform gels around the cellulose-hemicellulose fibrils which allow forsideways fibril slippage and heavily influence the cell wall porosityand thickness (30). Lignin, an amorphous phenolic polymer which isdeposited within the carbohydrate matrix of the cell wall in the finalstages of cell differentiation, provides structural integrity byenabling load transfer to the cellulose microfibrils, while alsoproviding resistance to external pathogens (53).

Accordingly, in some embodiments of biocomposite of the disclosure andrelated compositions, methods and systems, plant cell walls ofphotosynthetic eukaryotes of the kingdom Plantae according to thedisclosure are primarily composed of semi-crystalline cellulosemicrofibrils, which have remarkable mechanical properties (Young'smodulus up to 220 GPa, and tensile strength up to 7.7 GPa, depending oncrystallinity and physical characteristics (Gibson, 2012)) and areimmersed in a complex matrix of amorphous hemicelluloses, lignins andpectins. Model of the structure and functions of plant cell wallcomponents still remains to be refined (Cosgrove, 2005, 2014).

Exemplary plant cells present in a photosynthetic eukaryote of thekingdom Plantae comprises a cell from Chlorokybophyta,Mesostigmatophyta, Spirotaenia, Chlorobionta, Chlorophyta,Streptobionta, Klebsormidiophyceae, Charophyta (stoneworts),Chaetosphaeridiales, Coleochaetophyta, Zygnematophyta, Embryophyta (landplants), Marchantiophyta (liverworts), Bryophyta (mosses),Anthocerotophyta (hornworts), Horneophyta, Aglaophyta, and Tracheophyta(vascular plants).

In particular exemplary trachephyta plant cells comprise plant cells ofthe Nicotiana genus in Solanaceae family, includes Nicotiana attenuate(coyote tobacco), Nicotiana obtusifolia, Nicotiana acuminate, NicotianaAfricana, Nicotiana alata, Nicotiana benthamiana, Nicotiana clevelandii,Nicotiana glauca, Nicotiana glutinosa, Nicotiana langsdorffii, Nicotianalongiflora, Nicotiana occidentalis, Nicotiana otophora, Nicotianaplumbaginifolia, Nicotiana quadrivalvis, Nicotiana rustica, Nicotianasuaveolens, Nicotiana sylvestris, Nicotiana tabacum, and Nicotianatomentosiformis.

Exemplary trachephyta plant cells further comprise plant cells of theArabidopsis genus of the Brassicaceae family, comprising Arabidopsisarenicola, Arabidopsis arenosa (L.) Lawalrée, Arabidopsis croatica(Schott), Arabidopsis halleri (L.), Arabidopsis lyrata (L), Arabidopsisneglecta (Schultes) Arabidopsis pedemontana (Boiss.)Arabidopsis suecica(Fries) Norrlin, Meddel. And Arabidopsis thaliana (L.) Heynh.

In algae cells in accordance with the disclosure the native compositionof the of the cell walls is more diverse than in plants and isspecies-dependent. Cell walls of algae cells are composite in naturehaving a crystalline component and an amorphous component. In particularcell walls of algae, have glycoproteins and carbohydrates as the mainglucose-based components crystalline biopolymer, rigid and of glucosicnature forming a crystalline, which in some instances can be in afibrous form, (e.g. as cellulose). Cell wall biopolymers of algae formone or more distinct layers (possibly at least 2), identifiable by TEM.

Accordingly, the primary cell wall of photosynthetic eukaryotes of thegroup Algae is typically composed of glycoproteins and polysaccharidessuch as alginate, starch carrageenan and agar. Algae cell walls areextremely diverse and have very different arrangements among differentspecies. For example, the cell wall of Chlamydomonas reinhardtii is amultilayered extracellular matrix, cellulose-deficient, composed ofcarbohydrates and 20-25 polypeptides. Other algae also have amultilayered cell wall with several distinct layers (e.g. at least 3 or5 layers depending on the species), some of which form a highlycrystalline lattice comprised of glycoprotein subunits. The outer layercan be fibrous. The red algae possess complex composite cell walls madeof cellulose, xylan or mannan fibrils and extensive matrixpolysaccharides including the economically important carrageenan andagar.

In some algae cells, the cell wall can be thin (e.g. 30 nm at minimum-),contain no cellulose and be comprised of carbohydrates and severalhydroxyproline-rich glycoproteins which are cross-linked to shortoligosaccharides. In those algae the crystalline lattice formed fromhydroxyproline-rich glycoproteins. The same crystalline component isfound for example at least in 15 green algae species belonging to theVolvocales, Chlorococcales, Codiolales, Desmidiales and Zygnematales,(see references [73] and (74)). In some green algae with differentglycoprotein crystalline structures (see reference 75). Another algae'sspecies, Botryococcusis composed of a fibrous cell wall layer of uniformthickness (50 nm) In other algae the polysaccharide cell walls compriseagarose (agar) and carrageenan are sulfated galactans (in red seaweeds).In some algae cell wall, the polysaccharide is comprised of therepeating disaccharide 3-β-D-Galp-1-4-3,6-anhydro-α-L-Galp-1 unit.Alginic acid, a linear polysaccharide composed of 1-4-linked β-D-ManAand its C-5 epimer 1-4-linked α-L-GulA, is obtained from various speciesof brown seaweed. (see: Chapter 24 reference 76))

Exemplary Alga eukaryote organism comprise a cell from Archaeplastida,Plantae/green algae, Mesostigmatophyceae, Chlorokybophyceae,Chlorophyta, Charophyta, Rhodophyta (red algae), Glaucophyta, Rhizaria,Excavata, Chlorarachniophytes, Euglenids, Chromista, Alveolata,Heterokonts, Bacillariophyceae (Diatoms), Axodines, Bolidomonas,Eustigmatophyceae, Phaeophyceae (brown algae), Chrysophyceae (goldenalgae), Raphidophyceae, Synurophyceae Xanthophyceae (yellow-greenalgae), Cryptophyta, Dinoflagellata Haptophyta and Chlamydomonas.

In particular, exemplary Chlamydomonas algae suitable for thebiocomposite of the disclosure that can be found in stagnant water andon damp soil, in freshwater, seawater, comprise Chlamydomonas includesSpecies: C. aalesundensis-C. abbreviata Chlamydomonas acidophila,Chlamydomonas caudata Wille, Chlamydomonas ehrenbergii Gorozhankin,Chlamydomonas elegans Chlamydomonas moewusii, Chlamydomonas nivalis,Chlamydomonas ovoidae and Chlamydomonas reinhardtii.

In plant cells in accordance with the disclosure the native compositionof the plant cell primary and possibly secondary walls, as well as thearrangement of the different components within the cell wall and on thehierarchical organization of the cells at the microscale are the primaryresponsible for the mechanical properties of any biomaterials based onthe plant cells. (Gibson and Ashby, 1997; Gibson, 2012).

In a plant cell according to the disclosure the composition of cellwalls varies between species and may depend on cell type anddevelopmental stage. Accordingly, in biocomposites according to thepresent disclosure, by controlling the native composition of plant cellwalls, organization of the main components in the cell walls and varyingthe cellular microstructure, plants can exhibit this remarkable range ofmechanical properties of biomaterials based on plant cells in the senseof the disclosure.

In some embodiments, the biocomposite of the present disclosure comprisea mixture of plant and algae cells from various sources and feedstockbiomass, preferably from renewable and/or sustainable sources.

In some embodiments, the biocomposite of the present disclosure compriseonly plant cells. from various sources and feedstock biomass, preferablyfrom renewable and/or sustainable sources.

In some embodiments, the biocomposite of the present disclosure compriseonly plant cells from various sources and feedstock biomass, preferablyfrom renewable and/or sustainable sources.

A biocomposite according to the disclosure (herein also indicated as“AW” and “BC”) comprises compacted plant and/or algae cells having awater content of less than 15 wt %.

In particular in some embodiments of the biocomposite as describedherein, the compacted plant and/or algae cells have a water contentequal to or less than 12 wt %.

In particular in some embodiments of the biocomposite as describedherein, the compacted plant and/or algae cells have a water contentequal to or less than 10 wt %.

In particular in some embodiments of the biocomposite as describedherein, the compacted plant and/or algae cells have a water contentranging from 6 wt %. to 8 wt %

In some embodiments of the biocomposite as described herein, thecompacted plant and/or algae cells have a water content equal to or lessthan 2.0 wt %.

In some embodiments of the biocomposite as described herein, thecompacted plant and/or algae cells have a water content equal to or lessthan 1.0 wt %.

In some embodiments of the biocomposite as described herein, thecompacted plant and/or algae cells have a water content equal to or lessthan 0.1 wt %.

Accordingly, biocomposite of the instant disclosure can have dry densityranging from 500-1500 kg/m3, in combination with a water content between0.1-15 wt %.

The term “density” as used herein indicates the density of thebiocomposite taken in the dry state. The biocomposite mass is commonly amixture of air, water and compacted plant and/or algae cells solids. Thedry density refers to compacted plant and/or algae cells solids. The drydensity can be calculated using the expression as below,ρd=Ms/Vtwherein ρd indicates the dry density, Ms is mass of the soil solids andVT is total soil solid volume.

In some embodiments, biocomposite of the instant disclosure can have adry density ranging from of 900-1100 kg/m³, in particular, when thebiocomposite comprises plant cells.

In some embodiments, biocomposite of the instant disclosure can have adry density ranging from of 900-1000 kg/m³, in particular, when thebiocomposite comprises algae cells.

A biocomposite according to the disclosure is further poreless or haspores with a diameter of less than 10 m.

The term “pore” as described herein indicates a cavity that has a volumeequivalent to that of a sphere having diameter of at least 1 nanometer.Accordingly a pore in the sense of the disclosure is a void that remainsunfilled with polymers and other components in a composite material.Pores can affect the mechanical properties and lifespan of thecomposite. They degrade mainly the matrix-dominated properties such asinterlaminar shear strength, longitudinal compressive strength, andtransverse tensile strength. Pores contribute to the substance'sporosity and can be detected through Scanning Electron Microscopy (SEM)imaging, Transmission Electron (TEM) imaging, X-ray tomography andadditional methods identifiable by a skilled person. Accordingly, theterm as used herein the term “poreless” refers to absence of a cavitythat has a volume equivalent to that of a sphere having diameter of atleast 1 nanometer as detectable with these detection techniques.

In embodiments of the biocomposite as described herein, the compactedplant and/or algae cells have pores with a diameter of less than 10 μm.

Preferably in embodiments of the biocomposite as described herein, thecompacted plant and/or algae cells have pores with a diameter of lessthan 5 μm.

More preferably in embodiments of the biocomposite as described herein,the compacted plant and/or algae cells have pores with a diameter ofless than 3 μm.

Even more preferably in embodiments of the biocompo site as describedherein, the compacted plant and/or algae cells have pores with adiameter of less than 2 μm.

Most preferably in embodiments of the biocomposite as described herein,the compacted plant and/or algae cells have pores with a diameter of 1μm or less.

In biocomposite of the instant disclosure the compacted plant and/oralgae cells form lamellae having a thickness of 20 nm to 5 μm.

The term “lamella” as used herein indicates a thin layer of materialhaving a same structural feature. Accordingly, lamellar structures canbe fine layers of a same material or alternating and/or in seriesbetween different materials in various configuration. For example, alamellar structure can refer to collections of fine sheets of materialheld adjacent to one another (e.g. in a gill-shaped structure) or oneabove the other (a stacked configuration), possibly with fluid inbetween though sometimes simply a set of ‘welded’ plates. Lamellae canbe produced by chemical effects (as in eutectic solidification),biological means, or a deliberate process of lamination, such as patternwelding. Lamellae can also describe the layers of atoms in the crystallattice of a material such as a metal.

In biocomposite of the instant disclosure, each lamella comprises a thinlayer of collapsed plant and/or algae cell walls forming asemi-crystalline structure in which a crystalline component of a glucosebased biopolymer (typically a polysaccharide and/or glycoprotein) of theplant and/or algae cell wall, is immersed in an amorphous matrix ofother sugar-based and/or phenol-based biopolymers, typicallycarbohydrate, polysaccharide or oligosaccharide, phenolic biopolymerics,of the plant and/or algae cell wall. The semi-crystalline structure canfurther comprise additional material and in particular, additionalmaterial from the plant and/or algae cell and/or cell wall, as will beunderstood by a skilled person.

As used herein, “crystalline” when used in connection with a biopolymerrefers to a structural characteristic of a biopolymer which is arrangedperiodically in an ordered microscopic structure, forming a crystallattice that extends in all direction.

Conversely the wording “amorphous” when used in connection with abiopolymer indicates a biopolymer that lacks the long-range order thatis characteristic of a crystal.

Accordingly, a “crystalline” biopolymer as used herein indicates abiopolymer that has a crystalline structure or if the biopolymer canhave both amorphous and crystalline structure the percentage of thebiopolymer that is present in a crystalline form when the biopolymer canalso be present in both amorphous form. The crystallinity can bedetermined experimentally by X-ray powder diffraction pattern based onmeasurement of the intensity of a diffraction peak using crystallineform and amorphous forms as control samples.

The wording “semi crystalline” material as used herein is a substancethat has less than 100% of crystallinity and higher than 0% of amorphouscontent, including 50% each of crystallinity and amorphous contents. Bythe term “rigid semi crystalline,” it is meant that a semi-crystallinematerial has a high degree of order and periodicity which leads tohigher stiffness and strength compared to other non-crystallinebiopolymers in the cell wall composite, it is therefore the load-bearingor reinforcing component.

In some embodiments, the crystalline component comprises polysaccharidefibrils in an amorphous matrix of sugar based and/or phenol-basedbiopolymer from cell walls of the compacted plant and/or algae cells. Inparticular in biocomposite of the present disclosure, wherein the plantand/or algae cells comprise a plant cell, crystalline componentcomprises polysaccharide fibrils, and in particular cellulose fibrils.

The term “polysaccharide” as used herein indicates any polysaccharidethat comprises at least three monosaccharide monomeric moieties.Exemplary monosaccharide monomeric moiety includes D-glucose units,D-mannopyranuronose, L-gulopyranuronose, and L-gulopyranuronic acid.

The term “fibril” as used herein indicate an linear aggregate composedof linear biopolymers, characterized by rod-like structures with athickness of 1 to 10 nm and a high length-to-diameter ratios of at least2 to 1, preferably at least 10 to 1, more preferable at least 100 to 1.Fibrils can spontaneously arrange into helical structures. Fibrils areusually found alone but rather are parts of greater hierarchicalstructures commonly found in biological systems. Differences instructure between fibrils of different origin is typically determined byx-ray diffraction. A scanning electron microscope (SEM) can be used toobserve specific details on larger fibril species.

The term “fiber” as used herein indicates rod shaped substance that hasa length at least twice longer than its width or diameter havingthickness between 10-50 nanometers and forming micro to milli-scalestructures. As used herein, a fiber can made of a plurality of fibrilsarranged in parallel and in contact with each other.

Polysaccharide fibrils in the sense of the disclosure comprise anypolysaccharide that comprises at least three monosaccharide monomericmoieties aggregates as a rod structure. Exemplary polysaccharide fibrilincludes cellulose which comprises β(1→4) linked D-glucose units.Another exemplary polysaccharide fibril includes alginate fibril whichcomprises repeat sequences ofβ-D-mannopyranuronosyl-(1→4)-α-L-gulopyranuronosyl-(1→4)-α-L-gulopyranuronate.

In biocomposite of the present disclosure, wherein the plant and/oralgae cells comprise an algae cell, comprise e crystalline glucose basedbiopolymers, such as hydroxyproline-rich glycoproteins, polysaccharidefibrils such as cellulose, alginic acid or calcium alginate, abiopolymer containing or a polysaccharide of beta-1,3-xylan,beta-1,4-xylan, and/or beta-1,4-mannan, in particular wherein the plantand/or algae cells comprise an algae cell.

In biocomposite of the present disclosure crystalline biopolymers withina lamella are comprised within an amorphous matrix comprisingsugar-based biopolymers and/or phenol-based biopolymers from the cellwall of the plant and/or algae cells.

The term “amorphous matrix” as used herein indicates a solid that lacksa long range order and which serves to embed another solid substance.Accordingly an amorphous matrix is characterized by a highly irregularstructure compared with that of the crystalline component and can have agel-like consistency.

In biocomposite of the present disclosure, the composition of anamorphous material and is moreover known to vary considerably as afunction of the cell wall composition of the plant and/or algae cells ofthe disclosure.

In biocomposite of the present disclosure, the amorphous matrix can be apolysaccharide matrix which can optionally comprise phenol-basedbiopolymers such as lignin or lignols.

In particular, in biocomposite of the disclosure comprising a plantcell, the amorphous polysaccharide matrix can comprise hemicellulosesand pectin and optionally lignin.

In a biocomposite of the disclosure comprising an algae cell, theamorphous polysaccharide matrix can comprise hemicellulose, pectin,ligninol, carrageenan, agar, a polysaccharide or a biopolymer containingat least one of xylogalactoarabinan, glucuronoxylorhamnan,rhamnoxylogalactogalacturonan, and 3-deoxylxo-2-heptulosaric acid or anysulfated derivative thereof.

In some embodiments of the biocomposite as described herein, thepolysaccharide fibril comprises cellulose fibrils and the polysaccharideamorphous matrix comprises hemicellulose, pectin and optionally lignol,or any combination thereof.

In some embodiments of the biocomposite as described herein, thepolysaccharide fibril comprises includes alginate and the polysaccharideamorphous matrix comprises carrageenan an/or agar, and optionallycellulose, hemicellulose, pectin and/or any combination thereof.

In embodiments of the biocomposite as described herein, thepolysaccharide fibril comprises cellulose in a polymorph of Iα, Iβ, IIor III or any combination thereof.

In some embodiments of the biocomposite as described herein, the fibrilscomprise cellulose in form of crystalline or semi-crystalline cellulosemicrofibril.

In some embodiments the biocomposite as described herein comprisescellulose in an amount ranging up to 30.0 wt %, possibly up to 15.0 wt %or up to 5 wt %.

In some embodiments, the biocomposite as described herein compriseshemicellulose in an amount up to 30%, possibly up to 20% up to 10 wt %or up to 5 wt %.

In some embodiments the biocomposite as described herein comprisespectin in an amount up to 30.0 wt %, possibly up to 10.0 wt % or up to 5wt %.

In some embodiments the biocomposite as described herein, compriseslignols in an amount up to 10.0 wt %, possibly from 5% to 10% orpossibly up to 5 wt %.

In some embodiments the biocomposite as described herein, wherein theplant and/or algae cells are from tobacco can comprise 15 wt %cellulose, 20 wt % hemicelluloses, 6.8 wt % pectins and 6.3 wt %lignols.

In biocomposite of the disclosure, each lamella independently has athickness of 100 nm to 5 μm.

In some embodiments of the biocomposite of the disclosure, lamellae canindependently have a thickness of 20 nm to 100 nm. In some of thoseembodiments, the plant and/or algae cell comprise or consist of algaecells.

In some embodiments of the biocomposite of the disclosure, lamellae canindependently have a thickness of 100 nm to 300 nm. In some of thoseembodiments, the plant and/or algae cell comprise or consist of plantcells with a primary cell wall and without a secondary cell wall.

In some embodiments of the biocomposite of the disclosure, lamellae canindependently have a thickness of 20 nm to 300 nm. In some of thoseembodiments, the plant and/or algae cell comprise algae cells plantcells with a primary cell wall and without a secondary cell wall.

In some embodiments of the biocomposite of the disclosure, lamellae canindependently have a thickness of 300 nm up 5μ. In some of thoseembodiments, the plant and/or algae cell comprise or consist of plantcells within one or more secondary cell wall.

In some embodiments of the biocomposite as described herein, lamellaecan independently have a thickness ranging from 50 nm to 300 nm.

In some embodiments of the biocomposite as described herein, lamellaecan independently have a thickness ranging from 150 nm to 250 nm.

In some embodiments of the biocomposite as described herein, lamellaecan independently have a thickness ranging from 175 nm to 200 nm.

In general in biocomposite of the instant disclosure a hierarchical andanisotropic lamellar microstructure with compacted cells are alignedalong a plane. In the nanoscale, at the sub-cellular level, a fibrillarmulti-lamellated structure is also observed.

In particular biocomposites of the disclosure the lamellae can bedetected by microscopy images which show folded lamellae, derived fromcompacting cells. In particular in biocomposites of the disclosuremicroscopy will show the layers formed by cell walls only found in plantcells/algae in this unique configuration, in which polysaccharide fibers(e.g. cellulose fibers) are included in the amorphous matrix ofpolysaccharides (see e.g. Example 5 and FIG. 7 panel (A)). Thearrangement of fibrils in the matrix can vary as well as theirdistancing within a lamella and the distancing between lamellae, whichwould have the polysaccharide fibrils (e.g. rigid semi-crystallinecellulose) t dispersed in an amorphous matrix.

In some embodiments, in a lamella of the biocomposite herein described,the semi-crystalline structure are disposed on a plane.

In some embodiments, in a lamella of the biocomposite herein described,at least 5% of the glucose based biopolymers of the crystallinecomponent are aligned in parallel, In some of those embodiments theglucose based biopolymers comprises fibrils of a polysaccharide such ascellulose.

In some embodiments, in a lamella of the biocomposite herein describedat least 50% of the glucose based biopolymers of the crystallinecomponent are aligned in parallel. In some of those embodiments theglucose based biopolymer comprises fibrils of a polysaccharide such ascellulose.

In some embodiments, in a lamella of the biocomposite herein describedat least 90% of the glucose based biopolymers of the crystallinecomponent are aligned in parallel. In some of those embodiments theglucose based biopolymers comprises fibrils of a polysaccharide such ascellulose.

In some embodiments, in a lamella of the biocomposite herein describedwherein the glucose based biopolymers of the crystalline componentcomprises fibrils the fibril has a diameter ranging from 1 to 30 nm. Insome of those embodiments, the biopolymer is a polysaccharide and inparticular cellulose.

In embodiments of the biocomposite as described herein, thesemi-crystalline polysaccharide wherein the glucose based biopolymers ofthe crystalline component comprises fibrils the fibril is bound in abiopolymer matrix.

In some embodiments, the biocomposites of the disclosure capitalizes onthe plant cell's ability to synthesize intricate multi-lamellatedstructures of cellulose, hemicellulose, lignin and pectin in their cellwalls. The use of different cell cultures and/or genetically modifiedspecies allows the fabrication of materials with significantly alteredproperties.

In some embodiments from chemical and structural characterizations thebiocomposites of the disclosure is composed of the native primary cellwall components: a heterogeneous multi-lamellated mixture ofsemicrystalline cellulose fibrils, in an amorphous network ofheteropolysaccharides (hemicelluloses and pectin) and phenolic compoundswhich are the lignin precursors (monolignols).

In the present disclosure, undifferentiated tobacco cells are used as anexemplary and representative model system to provide representativeexamples of biocomposites of the disclosure and related compositions,methods and systems. These cells multiply rapidly (ca. a factor of80-100 every 7 days (22), can be used to produce materials in-situ, andcan be cold pressed in molds of different shapes and sizes. The hereindescribed materials retain the native plant cell wall compositionnaturally secreted by growing plant cells, to achieve mechanicalperformance comparable to structural and engineered woods, and polymers.The microstructure, composition and mechanical properties of theproduced panels are characterized as described in the Examples section.It is shown that the incorporation of filler additives allowsimprovement of the material's performance and expands theirfunctionalities, for example creating magnetic and electricallyconductive materials. The results obtained for tobacco cells have beenconfirmed by experiments in Arabidopsis and in the algae Chlamydomonasreinhardtii which provides a representative example of algae cellssuitable for the preparation of the biocomposite of the instantdisclosure.

In some embodiments, based on the results obtained in the representativeexamples of Tobacco and Chlamydomonas reinhardtii, the biocomposite ofthe disclosure can have a flexural modulus 0.1-7 GPa, modulus of rupture5-95 MPa, Young's modulus 1.5-5 GPa, tensile strength 9-25 MPa, and/orcompressive modulus 0.04-1.5 GPa. In some of those embodiments, thebiocomposite comprises plant cells from tobacco cells, cells fromArabidopsis and/or Chlamydomonas reinhardtii.

Biocomposite according to the present disclosure can be used asbiodegradable environmentally friendly alternative to non-degradablematerials, which typically survive in landfills. In particular,biocomposite of the present disclosure of 20-50 mm³ in volumedisintegrates in water in a time ranging from 24 hrs to 1 week andflakes between 0.1-2 mg in weight are expected degrade in soil in a timeranging from 10 weeks to 15 weeks, possibly between 12 to 14 weeksdepending on plant and/or algae cells comprised within the biocomposite.Accordingly, biocomposite of the present disclosure can be disintegratedor degraded by putting it in soil or water.

Biocomposite according to the present disclosure can have a controlledlongevity if placed under controlled humidity, in particular, in anindoor environment can remain substantially as fabricated for timeperiod of at least three years or more depending on the features of thebiocomposite. Longevity biocomposite of the instant disclosure can beincreased by addition of a polymer coating as will be understood by askilled person upon reading of the present disclosure.

In some embodiments of the biocomposite as described herein, thebiocomposite further comprises at least one additive The term “additive”as used herein refers any material is comprised within the plurality ofplant cells in a biocomposite according to the instant disclosure. Anadditive can for example be a filler, or a binder.

The term “filler” as used herein refers to any solid material that aremixed with dehydrated cells in the biocomposite and which retains aboundary of the solid material in the biocomposite. Exemplary fillerincludes natural or artificial fibers, and in particular plant fibersuch as flax fiber and inorganic particulate.

The term “inorganic particulate” as used herein refers to a solidmaterial that are equal to or less than 1 centimeter in a longestdimension that does not contain carbon-hydrogen bond. Exemplary fillersinclude carbon fiber, graphene, halloysite, montmorillonite nanoclay,iron oxide nanoparticle. The filler can have amorphous or crystalline.The inorganic particulate can have a regular or irregular shape. Regularshape includes, for example, sphere, prolate ellipsoid, oblateellipsoid, rod or cylinder, cube, or flake. The inorganic particulatecan, for example, have a longest span of 1 nanometer to 1 centimeter, 1micron to 1 millimeter, or 10 microns to 100 microns. For example, aniron oxide nanoparticle can, for example, shape as a sphere with adiameter of 1 to 100 nanometers.

The term “longest span” refers to the greatest distance between any twopoints in a solid of any shape. For example, a spherical particle wouldhave a longest span equal to a diameter of the spherical particle. A rodshape particulate would have a longest span equal to the longitudinaldimension of the rod. A fiber component of a plant cell can, forexample, have a longest span of 100 micron to 10 centimeters.

The term “binder” as used herein refers to any material that is mixedwith dehydrated cells in a biocomposite and which permeates continuouslythroughout the solid surfaces in the biocomposite.

Different filler particles expand the biocomposites' property space.Elastic modulus can be plotted as a function of density of differentplant-based biocomposites as described in the Examples section andrelated illustration where plots with respect to, pure cell matrix (BC),biocomposites containing various amounts of CF, halloysite andmontmorillonite nanoclays (NC) and graphene (G). are shown Theirproperties lie at the intersection of natural cellular materials,including “wood products”, and commercial plastics (see Example 12)presenting elastic moduli spanning over one order of magnitude as willbe understood by a skilled person upon reading of the presentdisclosure.

Filler additives also endow new functionalities, such as electricalconductivity or magnetic properties. The electrical conductivity ofplant cell/CF composites, for example, can be tuned varying the CFcontent in which the IV plots of biocomposites containing 1 and 20 wt %of CF illustrate the effective tuning of electrical conductivity from2.25×10⁻⁷ S/m to 2.2×10⁻³ S/m. (see Example 12).

In some embodiments wherein a filler is included in the biocompositematerial of the disclosure biocomposites can include up to 40 wt %filler additives in amount and type selected depending on the desiredmechanical properties of the resulting biocomposite.

In some embodiments, for example, wherein a filler is included in thebiocomposite material of the disclosure, a strength enhancement of thebiocomposite is obtained, for example in connection with addition ofabout 12% at 5 wt % of carbon fibers, followed by a monotonic decreaseat higher filler loadings. This is a behavior typically observed inpolymer composite materials, in which at low filler concentrations amore efficient filler dispersion can be obtained, thereby enablingsuccessful load transfer between the two components. In contrast, athigher concentrations, the filler particles aggregate, thus contributingto an inhomogeneous stress distribution upon loading which leads tooverall inferior mechanical performance (Roumeli et al., 2014), In someof these embodiments, the plant and/or algae cell is a tobacco cell andthe filler is a carbon fiber.

Similarly, the addition of 13.5 wt % iron oxide nanoparticles (IN) inthe plant cell matrix conveys ferro-magnetic properties, which allow thebiocomposite to support more than five times, or six times its weightwhen attracted by a magnet. (see Example 12). In some of theseembodiments, the plant and/or algae cell is a tobacco cell and thefiller is iron.

In some embodiments of the biocomposite as described herein, the atleast one filler is a natural filler is comprised in biocomposite inaddition to the plurality of plant cells.

In embodiments of the biocomposite as described herein, the naturalfiller is a plant fiber.

In embodiments of the biocomposite as described herein, the plant filleris a flax fiber embedded in the dehydrated cell.

In embodiments of the biocomposite as described herein, the naturalfiller is a biomass.

In embodiments of the biocomposite as described herein, the naturalfiller is a silk fiber.

In embodiments of the biocomposite as described herein, the at least onefiller is selected from the group consisting of carbon fiber, graphene,halloysite, montmorillonite nanoclay, and iron oxide nanoparticle or anycombination thereof.

In embodiments of the biocomposite as described herein, the at least onefiller comprises iron oxide nanoparticle in an amount of 5.0-25.0 wt %.

In embodiments of the biocomposite as described herein, the at least onefiller comprises iron oxide nanoparticle in an amount of 10.0-15.0 wt %.

In embodiments of the biocomposite as described herein, the biocompositefurther comprises at least one binder.

In embodiments of the biocomposite as described herein, the at least onebinder is selected from the group consisting of alginate, chitosan,pectin, lignin and cellulose, or any combination thereof.

In embodiments of the biocomposite as described herein, the alginate,chitosan, pectin, lignin and cellulose are each present in an amount of0.1 to 10.0 wt %.

In embodiments of the biocomposite as described herein, the biocompositeis part of a product comprising makeup container, food container, foodpackaging material, toy packaging material, shipping packaging material,cafeteria trays, furniture, clothes hanger, and multi-use packaging.

In some embodiments of the biocomposite as described herein, thebiocomposite can be coated with one or more materials to provideadditional properties to the biocomposite material. In particular,hydrophobic material is coated with a hydrophobic polymer to impartwater resistance to the biocomposite.

The term “hydrophobic polymer” refers to any polymeric organic materialthat has a static water contact angle θ that is greater than 90°(Kock-Yee Law 2014).

In particular when not requiring waterproof material where it isdesired, such as application of a polymer coating on surface of thedevice made of the biocomposite material herein described.

In some embodiments of the biocomposite as described herein, thehydrophobic polymer can be selected from poly(dimethylsiloxane),polymethylmethacrylate, polyethylmethacrylate, polymethylacrylate,polyethylacrylate, polybutylacrylate, polyethylene, polypropylene, EPDMrubber, polyvinyl chloride, polyvinylfluoride, polyvinylidene fluoride,polytetrafluoroethylene, perfluoroalkoxy polymer, poly(butadiene),poly(isoprene), ethylene-butene copolymer, poly(norbornene),poly(siloxane), polyester, and polyurethane, epoxy resin or anycombination thereof.

Biocomposites herein described are provided with a fabrication methodaccording to the present disclosure which allows formation of thelamellar configuration of the biocomposite from plant and/or algae cellsleveraging the naturally occurring composite nature of the cell wall ofplant and/or algae cells, while minimizing pores formation and/ordimension of the pores. In particular in the fabrication method of thepresent disclosure the cell walls of the plant and/or algae cell arecompacted in a semi-crystalline structure comprising biopolymers of thecell wall according to a process that minimizes the formation of poresas will be understood by a skilled person upon reading of the presentdisclosure.

The fabrication method of the present disclosure comprises compactingalong a plane a cultured biomass comprising cultured plant cells from asuspension culture having a water content of at least 10 wt %.

The wording “cultured cells” as used herein indicates cells undercontrolled conditions, generally outside their natural environment.Accordingly, “cultured plant cells” indicates plant cells undercontrolled conditions outside their natural environment. Typically,cultured plant cells are isolated from a feedstock biomass and/or fromliving organisms comprising plant cells according to the presentdisclosure, and subsequently be maintained under controlled cultureconditions.

The term “biomass” as used in the present disclosure indicates materialof biological origin. The term “feedstock biomass” as used hereinindicates a raw and/or, unprocessed material of biological origins usedas a basic material. In particular feedstock biomass can be used toproduce goods, finished products, energy, or intermediate materials thatare feedstock for future finished products, or primary commodity,production of energy (electricity or heat), or in various industrialprocesses as raw substance for a range of products. Feedstock biomasscomprising plant cells in the sense of the disclosure comprisespurposely grown energy crops (e.g. miscanthus, switchgrass), wood orforest residues, waste from food crops (wheat straw, bagasse),horticulture (yard waste), food processing (corn cobs) and additionalfeedstock biomass identifiable by a skilled person upon reading of thepresent disclosure.

In some embodiments, the feedstock biomass can be formed or comprisefrom agricultural waste and/or agricultural residues (e.g., corn stove,fibers and/or particles r), Algae, dedicated energy crops (e.g.,switchgrass, miscanthus, energy cane, sweet sorghum, high biomasssorghum, hybrid poplars, and shrub willows), forestry residues (e.g.,logging residues and forest thinning), waste streams and re-useablecarbon biosources (e.g., the non-recyclable organic portion of municipalsolid waste, biosolids, sludges, waste food, and manure slurries).

In embodiments of the instant disclosure, cells isolated from afeedstock biomass (e.g. from a plant tissue or from an algae) arecultured in a suspension culture. In particular, in some embodiments ofthe fabrication method herein described, a plant cells and/or algae cellcan be separated from a feedstock biomass and mixed with culture mediumto provide a plurality of cultured plant cells in a suspension culture.

The wording “suspension culture” or “cell suspension” as used hereinindicates a type of cell culture in which single cells or smallaggregates of cells (e.g. clumps up to 50 cells, possibly from 5 to 20cells depending on the specific type of cells) are allowed to functionand multiply in an agitated growth medium, thus forming a suspension.Suspension cultures are used in addition to so-called adherent cultures.The cells themselves can either be derived from homogenized tissue orfrom another type of culture.

In some embodiments of the fabrication methods of the presentdisclosure, after being harvested, cultured plant cells from asuspension culture are subsequently typically maintained undercontrolled conditions. These conditions vary for each cell type, butgenerally consist of a suitable vessel with a substrate or medium thatsupplies the essential nutrients (amino acids, carbohydrates, vitamins,minerals), growth factors, hormones, and gases (CO2, O2), and regulatesthe physio-chemical environment (pH buffer, osmotic pressure,temperature). Most cells require a surface or an artificial substrate(adherent or monolayer culture) whereas others can be grown freefloating in culture medium (suspension culture). The lifespan of mostcells is genetically determined, but some cell culturing cells have been“transformed” into immortal cells which will reproduce indefinitely ifthe optimal conditions are provided.

In embodiments of the fabrication methods of the present disclosure,cells from the suspension culture comprising plant and/or algae cells,are separated from the suspension culture (e.g. by filtration) toprovide a cultured biomass from the suspension culture, typicallycomprising single cells or small clumps of cells (e.g. up to 20 or 50cells). For example, cells can be separated from medium by vacuumfiltration (e.g. an Erlenmeyer flask with a filter paper with a mesh of1-25 micrometers connected to a vacuum pump.

In embodiments of the fabrication method herein described, the culturedbiomass of a cultured plant and/or algae cells separated from theculture medium is then prepared for further compression.

In particular, in embodiments of the fabrication method of the instantdisclosure, the cultured biomass compressing plant cells and/or or algaecells is compacted to eliminate unbound water in the biomass whileforming a lamellar structure minimizing presence and/or size of pores.In particular in fabrication method of the instant disclosure, thecompacting is performed by applying a pressure for a time and undercondition to control their dehydration and aggregation.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, fabrication method is described according to whichthe cultured biomass can be compressed inside a compression tool toobtain a condensed lamellar structure.

In particular, in fabrication method of the disclosure, the culturedbiomass is then compressed along a plane by continuously applying to thecultured biomass an increasing pressure perpendicular to the plane untilreaching an applied pressure corresponding to the turgor pressure of thecultured plant and/or algae cells to provide a compacted biomass.

The word “turgor” as used herein with respect to a plant cell indicatesthe state of turgidity and resulting rigidity of cells or tissues,typically due to the absorption of fluid. Accordingly any plant cells inthe sense of the disclosure has a turgor pressure, wherein the wording“turgor pressure” or “hydrostatic pressure” indicates the force withinthe cell that pushes the plasma membrane against the cell wall. Theturgor pressure, is defined as the pressure measured by a fluid,measured at a certain point within itself when at equilibrium.Generally, turgor pressure is caused by the osmotic flow of water andoccurs in the plant cells. The pressure exerted by the osmotic flow ofwater is also called turgidity. It is caused by the osmotic flow ofwater through a selectively permeable membrane. Osmotic flow of waterthrough a semipermeable membrane is when the water travels from an areawith a low-solute concentration, to one with a higher-soluteconcentration. In plants cells in the sense of the disclosure, thisentails the water moving from the low concentration solute outside thecell, into the cell's vacuole.

In embodiments of the method as described herein, the applied pressurecorresponding to the turgor pressure of the cultured plant and/or algaecells ranges from 0.1 to 5.0 MPa.

In embodiments of the method as described herein, the applied pressurecorresponding to the turgor pressure of the cultured plant and/or algaecells ranges from 0.5 to 1.5 MPa.

Typically, in embodiments of the method as described herein, the appliedpressure corresponding to the turgor pressure of the cultured plantand/or algae cells ranges between 0.8-1.0 MPa.

Typically, in embodiments of the fabrication method of the disclosurethe increasing pressure comprises a start pressure ranging from 5% to15% of the turgor pressure.

In embodiments of the method as described herein, the rate of increasingpressure is a constant ranging from 0.01 to 0.5 MPa/day.

In embodiments of the method as described herein, the rate of increasingpressure is a constant ranging from 0.05 to 0.15 MPa/day.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, the selected biomaterials are compressed at acontinuous and varying pressure to control their compaction andcorresponding dehydration over time. In particular the applied pressuresincreases over time (e.g. with a step function or linear increase) toprovide a compressed biomass.

In some embodiments continuously applying pressure can be performed tocontinuously apply a pressure increasing according to a step function.In preferred embodiments continuously applying pressure can be performedto continuously apply a pressure increasing linearly to minimize poresformation and diameter.

In some embodiments, the continuously applying pressure can be performedby applying continuous pressure up to 0.8 MPA increasing the pressure of0.1 MPa per day up to 0.8 MPa. In some of these embodiments, theapplying can be performed from a time ranging from 6 to 8 days, or from5 to 7 days. In some embodiments, the continuously applying pressure canbe performed by applying continuous pressure for 4-5 days.

In the fabrication method of the present disclosure typically acompressed biomass is obtained after a mass loss of at least 50% andpossibly at least 80% with respect to the cultured biomass beforecompressing. Mass loss can be detected by weighing and the culturedbiomass and weighing the compressed biomass and compare the culturedbiomass and the compressed biomass weight as will be understood by askilled person.

In embodiments of the fabrication methods herein described, thecompacting further comprises drying the compressed biomass by at leastone of applying to the compressed biomass a drying pressure and/orapplying a drying temperature to the compressed biomass to obtain abiocomposite material having water content of 0.1-15 wt %, a mass lossof 80-99% and/or a dry density of 500-1500 kg/m3 having the features ofthe biocomposite of the instant disclosure.

, In preferred embodiments, the drying pressure can be appliedcontinuously. In preferred embodiments, the drying pressure can beapplied lineralry and more preferably linearly and continuously. In someembodiments, the drying pressure can be the applied pressurecorresponding to the turgor pressure of the cultured plant and/or algaecells. In some embodiments, the drying pressure can be higher than theapplied pressure corresponding to the turgor pressure of the culturedplant and/or algae cells (e.g. two or three times higher). In someembodiments the drying pressure can be >50% of the turgor pressuredepending on the plant and/or algae of the compressed biomass. In someembodiments a drying pressure up to 5 MPa can be applied to compressedbiomass in the sense of the disclosure.

In some embodiments applying to the compressed biomass the appliedpressure is performed to obtain: a biocomposite material having a watercontent of less than 12, wt %, less than 10 wt %, from 6 wt %, to 8 wt%, or less than 1 wt %.

In some embodiments applying to the compressed biomass the appliedpressure is performed to obtain: a biocomposite material having a massloss of 97 to 99% in particular when the plant and/or algae cells of thecompressed biomass comprise plant cells or to 85 to 90% mass loss inparticular when the plant and/or algae cells of the compressed biomasscomprise algae cells.

In some embodiments applying to the compressed biomass the appliedpressure is performed to obtain: a biocomposite material having a drydensity of 500-1500 kg/m3, 900 kg/m3 and 1100 kg/m3, or between 900kg/m3 to 1000 kg/m3 in particular when the plant and/or algae cells ofthe compressed biomass comprise algae cells.

In some of those embodiments applying to the compressed biomass thedrying pressure corresponding to the turgor pressure of the culturedplant and/or algae cells can be performed without drying for two weeksup 1 month to up to 2 month depending on the materials.

In some preferred embodiments, the compressed biomass undergotemperature cycles to control their dehydration over time. In thoseembodiments of the fabrication method as described herein, the methodcan further comprises applying a: drying temperature the compressedbiomass optionally after a first or second weight for a drying time.

In those embodiments of the method as described herein, the dryingtemperature can range from room temperature to a temperature lower thanthe degrading temperature of the material (typically below 110° C., 130°C. or 150° C.). Typically the drying can be performed with a temperatureranging from 20 to 100° C., and in particular, ranging from 40° C. to85° C. In some embodiments heating at temperature up to 60° C. may beapplied to accelerate the dehydration process.

In embodiments of the method as described herein, the drying time rangesfrom 0.1 to 480 hours.

In embodiments of the method as described herein, the drying time rangesfrom 24 to 72 hours.

In particular, in some embodiments, the drying can be performed for of2-48 hours at a temperature ranging from 40° C. and 85° C., possibly at60° C.

In some embodiments of the fabrication methods of the instantdisclosure, the drying can be performed concurrently with thecompressing, wherein the selected biomaterials are compressed at varyingpressure to control their dehydration over time, where compression isapplied by a hydraulic hot press. In those embodiments, the temperaturecan be selected in combination with the applied pressure to allowincreasing compression until reaching an applied pressure correspondingto the turgor pressure of the plant and/or algae cells in the biomass.

In some embodiments of the fabrication methods of the instantdisclosure, a drying (dehydration) step can be performed on theextracted biomass before compressing, in addition or in the alternativeto the drying during the compression and/or after the compressing stepFor example, the process can be performed also using a hot-press inwhich high temperature contributes to the fabrication by speeding up theentire process in combination with the applied pressure.

An exemplary workflow of the method to provide biocomposite of thedisclosure is schematically illustrated in FIG. 1 .

Plant cells are harvested from a suspension culture (and compress themin a permeable mold, (See FIG. 1 Panels A to C)).

During compression, water diffuses through the plant cell wall and thecell volume is gradually reduced. Compression is performed to achieve adensified dehydrated structure.

In particular, in bottom-up fabrication method is illustrated in FIG. 1, plant cells or microalgae are harvested from a stable culture atspecific stages of their development, and subject them to a controlledcompression process. By compressing the cells inside a mold withincreasing pressure over time, water diffuses through the permeable cellwall, while the cell volume is gradually reduced.

When the cells reach a dry state, corresponding to an approximate 80-99%weight loss and in particular 98% weight loss, the process isterminated, and the resulting material is essentially a lamellar stackof compacted cell walls. (See FIG. 1 Panels D to F)). Exemplaryprocedures according to the approach schematically described in FIG. 1are described in Examples 3 and 11 of the instant disclosure.

The resulting biocomposite material of the present disclosure comprisenative plant cell wall composition, with cell wall biopolymers,primarily the native cellulose, hemicelluloses and pectins, maintainedafter cell dehydration and organized in a lamellar configuration.Characterization of microstructure, composition and mechanicalproperties of the produced material are disclosed, and which illustrateby example the obtained properties by incorporating synthetic filleradditives.

In some embodiments, starting from undifferentiated plant cells and/oralgae, the stiffness and strength of the produced biocomposites exceedthe corresponding performance of some commercial plastics of the samedensity, for example polystyrene, and low-density polyethylene, whilebeing entirely biodegradable. The mechanical properties of thesecomposites are tunable upon selection of fabrication conditions.

In some embodiments of the method of the disclosure, starting fromalgae, biodegradable composite materials are produced with mechanicalproperties similar to those of particle boards commercially used forready-to-assemble furniture. Upon controlled dehydration undercompression, algae are able to bind together fibers and fillingparticles. Algae have an enormous diversity and their large range ofdifferent compositions and structures can result in different mechanicalproperties and functions. They have a beneficial impact on theenvironment since they mitigate the carbon excess in the atmosphere byconsuming large quantities of CO2 during growth (e.g., to produce 1 Kgof microalgae, about 2 Kg of CO2 are typically sequestered [H. Herzog,D. Golomb, Encyclopedia of Energy 1: 1-11 (2004)]).

In an exemplary embodiment of the fabrication method of the disclosureplant cells and/or algae cells extracted from a suspension culture, in ahydrated form, with a water content above 10 wt %, are transferred in apermeable mold with a porosity at least 5%, and the material can beeither metal, alloy, polymer or ceramic. The biomass is then compactedby applying pressure with the mold. The applied pressure rate can beselected depending on the feedstock material. The application of heatduring compression can be used to reduce processing time. When thefeedstock material has reached a weight loss of more than 50% comparedto it's original state, the biocomposite can be extracted from the moldsand subjected to a final drying step at temperatures below 100° C. forat least 2 hr. Addition of additives id desired can be typicallyperformed by mixing the additive with the culture biomass to createhybrids before the transferring, or after providing the compressedbiomass at least for additives to be applied as a coating.

Drying of the cultured biomass separated from the suspension culture canalso be optionally performed if control of water content is desired.

In particular an exemplary fabrication method of the disclosure cancomprise the step of extracting a biomass comprising plant and/or algaecells in a hydrated state from a culture suspension, for example using avacuum filtering system to separate the culture biomass from growthmedia. The fabrication method can further comprise weighing thecollected biomass and optionally mixing and optionally mixing one ormore additives, the mixing either manual or with assistive equipment(e.g. stirrer, ultrasonication) are used to homogenize the mixture. Ifcontrol of water content is desired in view of the features of thebiocomposite, the biomass can be dried at this step in addition or inalternative to the mixing. Oven drying at temperatures below 100° C. forthe duration that is required to reach the water loss desired. Thefabrication method further comprises transferring the biomass with orwithout additives into the mold and applying pressure. Calibration ofthe pressure rate dictates the appropriate compression protocol; in anexemplary embodiment with a biomass comprising plant cells and theircomposites 0.1±0.05 MPa/day till 0.8 MPa was used, followed by 2 days at0.8 MPa. In the fabrication process the biocomposite material can beextracted from the mold after a mass loss of more that 50% is achieved,and dried for a minimum of 12 h at a temperature below 100° C.

An exemplary side by side illustration of parameters of the method ofthe disclosure performed with plant cells and algae cells is reported inTable 1 below

TABLE 1 Final retained solid mass Initial water Pressure Post-process(100% corresponds content rate dry to the starting wet biomass) plantcell 70 ± 10 wt % 0.1 ± 0.05 48 h at 60° C. 2 ± 1 wt % biomass MPa/daytill 0.8 MPa algae  70 ± 10 wt %* 0.1 ± 0.05 48 h at 60° C. 10 ± 5 wt % biomass MPa/day till 0.8 MPa

In an exemplary process to provide biocomposite from plant and/or algaecells in the sense of the disclosure, the pressure-time profile can becalibrated to achieve maximum product density. For example, inbiocomposite formed starting from plant cells the biomass used in ahydrated state of 60-80 wt % water can be provided with no pre-dryingstep to control or reduce water content. In order. to achieve a densityof 1100±100 kg/m³, a 0.1±0.05 MPa/day rate compression rate can beselected to a maximum of 0.8 MPa followed by a 2-day constantcompression at 0.8 MPa. A post-manufacturing drying step (48 h at 60° C.in a benchtop oven) can be applied and the retained until the dry samplemass was 2±1 wt % of the initial wet biomass. This last step does noteliminate all bound water and as all cellulose-based materials, storingthem in ambient conditions will result in atmospheric water uptake. Insome embodiments, absence of drying step and/or water uptake, followingfabrication do not affect mechanical properties of the resultingbiocomposite. In embodiments wherein the biocomposite is provided froman algae biomass, the final material obtained in outcome of thisexemplary fabrication process has a weight which can be 10±5 wt % of theinitial wet biomass and the density was 980±130 kg/m³.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, plant cells, algae, and biomass from agriculturalwaste (or combinations or the three) can be compressed inside a mold toobtain a condensed lamellar structure, wherein the selected algae areChlamydomonas, Desmodesmus, or a combination of the two or wherein theselected starting biomass comprises cyanobacteria.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, an approach to fabricate bulk, biological matrixcomposites based on plant cells, which grow and rapidly multiply insuspensions. To form lamellated panels of arbitrary geometries,harvested cells are dehydrated and compressed under controlledconditions, without the need for harsh chemical treatments or binders.The properties of these biocomposites are comparable to wood and woodproducts and superior to other self-growing natural composite materials.The biocomposites' properties can be further tuned varying thefabrication process. For example, filler particles can be integratedduring fabrication, to vary the mechanical response or introduce newfunctionalities. The process herein described introduces a novel classof biocomposite materials that can be extended to different plant cellsand scaled for large production.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, a natural bulk biocomposite material can be thuscreated based on plant cells. The method capitalizes on the plant cell'sability to synthesize intricate multi-lamellated structures ofcellulose, hemicellulose, lignin and pectin in their cell walls. The useof different cell cultures and/or genetically modified species allowsthe fabrication of materials with significantly altered properties.Similar fabrication approaches are contemplated for other biologicalsystems including but not limited to algae, and fungi that can providecomplex elements as building blocks for advanced composite biomaterials.

In some embodiments the methods to provide biocomposite of the instantdisclosure uses a natural biopolymer mixture as a matrix and incorporatefiller additives, which (i) introduces new properties/functions in thecomposites, and (ii) enables further tuning of the mechanicalperformance. The addition of different amounts of carbon fibers (CF),for example, changes the biocomposites' compressive modulus and strengthFor CF concentrations below 5 wt % there is a gradual improvement ofelastic modulus and strength, in the order of 20-25%, followed by adecrease for higher concentrations, as observed in polymer compositesbecause of fillers' aggregation (38) (see Example 12).

Accordingly, in some embodiments of the methods to provide biocompositeof the instant disclosure, biocomposite materials are provided in whichcyanobacteria are combined with dehydrated plant cells or algae (or acombination of them).

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite materials are provided in whichsynthetic and/or natural fillers (for example silk fibers, flax fibers,nanoclay) are immersed in a matrix made of dehydrated plant cells oralgae (or a combination of them).

In some embodiments of the methods to provide biocomposite of theinstant disclosure, tests can be performed to show that a controlleddehydration of microalgae can form a homogeneous matrix that is capableof binding together particles obtained by crushing or milling biomassfrom agricultural waste. These composites (APBs) already have mechanicalproperties similar to those of particle boards typically used forfurniture and kitchen applications (see preliminary results inattachments). Different strains of algae and different types ofagricultural waste can be used to tune the mechanical properties of thematerial. Different scaled up production processes are contemplatedwhich are suitable for various industry applications of the material.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite is provided in which alginate is mixedwith plant cells or algae (or a combination of the two) before theirdehydration following the methods as described herein, in which plantcells, algae, and biomass from agricultural waste (or combinations orthe three) are compressed inside a mold to obtain a condensed lamellarstructure, the selected bio-materials are compressed at varying pressureto control their dehydration over time, optionally in addition topressure, the selected bio-materials undergo temperature cycles tocontrol their dehydration over time.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite is provided in which chitosan is mixedwith plant cells or algae (or a combination of the two) before theirdehydration following the methods as described herein, in which plantcells, algae, and biomass from agricultural waste (or combinations orthe three) are compressed inside a mold to obtain a condensed lamellarstructure, the selected bio-materials are compressed at varying pressureto control their dehydration over time, optionally in addition topressure, the selected bio-materials undergo temperature cycles tocontrol their dehydration over time.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite is provided in which pectin is mixedwith plant cells or algae (or a combination of the two) before theirdehydration following the methods as described herein, in which plantcells, algae, and biomass from agricultural waste (or combinations orthe three) are compressed inside a mold to obtain a condensed lamellarstructure, the selected bio-materials are compressed at varying pressureto control their dehydration over time, optionally in addition topressure, the selected bio-materials undergo temperature cycles tocontrol their dehydration over time.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite is provided herein in which lignin ismixed with plant cells or algae (or a combination of the two) beforetheir dehydration following the methods as described herein, in whichplant cells, algae, and biomass from agricultural waste (or combinationsor the three) are compressed inside a mold to obtain a condensedlamellar structure, the selected bio-materials are compressed at varyingpressure to control their dehydration over time, optionally in additionto pressure, the selected bio-materials undergo temperature cycles tocontrol their dehydration over time.

In some embodiments of the methods to provide biocomposite of theinstant disclosure, biocomposite is provided in which cellulose is mixedwith plant cells or algae (or a combination of the two) before theirdehydration following the methods as described herein, in which plantcells, algae, and biomass from agricultural waste (or combinations orthe three) are compressed inside a mold to obtain a condensed lamellarstructure, the selected bio-materials are compressed at varying pressureto control their dehydration over time, optionally in addition topressure, the selected bio-materials undergo temperature cycles tocontrol their dehydration over time.

In general in biocomposite of the instant disclosure a hierarchical andanisotropic microstructure can be observed, in those comprised of purecells and in those with additives. The compression process compacts thecells and gives rise to a lamellar architecture in the microscale. Inall prepared biocomposites the compacted cells are aligned along thenormal to the compression plane. In the nanoscale, at the sub-cellularlevel, a fibrillar multi-lamellated structure is also observed. Inparticular, after the compression the cellulose fibrils are aligned inthe plane normal to compression.

In general in biocomposite of the instant disclosure, SEM images showthat with the present approach almost no pores/air voids are observed.Therefore, fabrication method and resulting biocomposite of the instantdisclosure porosity is reduced in order to improve mechanicalproperties, because that correlation is known to exist in all bulkmaterials between porosity and mechanical properties.

Plant cells, cultured plant cells, culture medium, biomass and acompression tools can be provided as part a system to provide abiocomposite herein described, the system comprising a cultured biomasscomprising cultured plant and/or algae cells from a suspension culturehaving a water content of at least 10 wt %, and a compression tool.

In embodiments of the biocomposite fabrication system the mold isconfigured to interact with the cultured biomass in accordance with thefabrication methods herein described to provide a biocomposite materialhaving water content of 0.1-15 wt %, a mass loss from the culturedbiomass of 80-99% and/or a dry density of 500-1500 kg/m3.

As used herein, a “compression tool” indicates any mechanism that canapply controlled varying levels of compression to a sample and allowwater to escape the sample (e.g. biocomposite panels). Examples includepresses, such as hydraulic, pneumatic, mechanical (e.g. screw), andelectromechanical (e.g. magnetically actuated) presses. The press can bea hot press which applies heat as well as pressure to the sample.

An example of a compression tool used for compression and dehydration isshown in FIG. 26 , in the form of a hydraulic hot press. A hydraulicactuator (2610) moves hydraulic pistons/rams (2620) to move a pressureplate (2630) to compress a sample (2650) against a bolster plate (2640).The pressure (controlled by the actuator (2610)) allows water to exit(2660) the sample. The pressure plate (2630) and/or bolster plate (2640)can be heated. The press can also be configured in other orientations,such as where the actuator, pistons, and pressure plate are below thesample and the pressure is applied upwards to a bolster plate above thesample. The sample can include a mold surrounding the material to bepressed, limiting lateral (with respect to the press plates) movement ofthe material. The press can include multiple plates stacked verticallysuch that multiple samples can be pressed at the same time. Thoseskilled in the art would understand the various press configurationsknown in the art.

In some embodiment, the compression tool is a compression mold.Preferably a compression mold is a porous more preferably an at least 5%porous mold, for example a 15% porous mold.

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can be used to address majorenvironmental concerns regarding composite materials include (i)sustainability of basic components, and (ii) recycling or degradationmethods of the significant production-related waste as well as after-usewaste (Mohanty et al., 2018; Huang et al., 2019). To date, the majorityof the produced composite materials waste is disposed of in landfills oris incinerated. Therefore, the urgent need to develop advanced compositematerials which combine biodegradability with sustainable maincomponents has emerged.

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can overcome challenges forthe all-bio-derived composites such as balancing the productioncost-performance relationships, improving durability and assessing thetrue environmental impact of manufacturing processing and post-usestrategies (Mohanty et al., 2018).

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can be used to providesustainable renewable material with mechanical properties superior tobiocomposite formed by yeast or mycelium with or without othercomponents to create biocomposites which have mechanical performancecharacterized by elastic modulus <0.6 GPa, strength <10 MPa.

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can be used to plan asustainable future reducing the impact the environment taken in term ofCO2 emissions that play a critical role in the rapid climate changes.

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can be used to providerenewable resources with reduced lifecycle environmental impacts withoutneed to rely on the use of polyolefin binders and matrixes, which arenot biodegradable or rely on the pulp and paper industry.

In some embodiments, biocomposites of the present disclosure andrelated, compositions, methods and systems, can be used to produce woodand plastic substitutes using algae and agricultural waste. Thebiomaterials as disclosed are biodegradable and can be directly formedin thick layers or panels, without the need for binders or adhesives.These materials will have a significant impact on the environment sincethey will recycle and add value to waste material and sequester CO2 fromthe atmosphere during their production. To produce 1 kg of microalgae,1.83 kg of CO2 is typically consumed. Algae can grow on land, which isunsuitable for agriculture and in wastewater, which would mitigate theneed for expensive nutrients.

The biocomposites, methods and systems herein described are furtherillustrated in the following examples, which are provided by way ofillustration and are not intended to be limiting.

EXAMPLES

The biocomposites, methods and systems herein described are furtherillustrated in the following examples, which are provided by way ofillustration and are not intended to be limiting.

In particular, biocompo sites from tobacco cells were prepared with aporous compression mold in accordance with an exemplary method of thepresent disclosure and the resulting biocomposites were tested forstructural, compositional and mechanical properties.

A person skilled in the art will appreciate the applicability and thenecessary modifications to adapt the features described in detail in thepresent section, to additional biocomposites and related compositions,methods and systems according to embodiments of the present disclosure.

In particular a skilled person will understand how to adapt thespecific, materials, and methods used in the following examples toadditional materials, and methods identifiable in view of the instantdisclosure such as additional, plant cells, biomass, mediums,compression mold, fillers, culturing, compression and/or detectionprocesses in accordance with the present disclosure.

The exemplary biocomposites, compositions, methods and systemsexemplified in this section were prepared and analyzed using thefollowing materials and methods.

Feedstock materials The natural wood materials tested were Red Oak(Quercus rubra), Black Walnut (Juglans nigra), Yellow Poplar(Liriodendron tulipifera) and Sugar Pine (Pinus lambertiana) and werekindly provided by Caltech resources. The engineered wood samples werehardwood plywood and medium density untempered hardboard (MDF) providedby Caltech resources. The commercial plastics were low-densitypolyethylene, LDPE (King Plastic Corp, North Port, FL.), SIS-030E highimpact polystyrene, PS (Certene, Norwalk, CT) and Densetec CopolymerPolypropylene, PP (Polymer Industries, Henagar, AL).

Cell staining: Cells were stained for cellulose in a 1% solution ofalcian blue in 3% acetic acid (MilliporeSigma, St. Louis, MO). Thestaining for pectin was performed using a 0.01% (v/v) ruthenium redsolution in water, supplemented with 0.1% (v/v) ammonia (MilliporeSigma,St. Louis, MO). Safranin O in a 1% solution was used to stain phenoliccompounds (MilliporeSigma, St. Louis, MO).

Microscopy Observations A Zeiss Axio Scope A1 (Zeiss, Oberkochen,Germany) was used for optical imaging of the untreated and stainedcells. Two-photon analysis of the safranin-stained cells was performedin a Zeiss LSM 710 confocal laser scanning microscope (Zeiss,Oberkochen, Germany). Image acquisition was implemented with a LCC-Apochromat 40×/1.1 W Korr M27 objective, at an excitation wavelengthof 488 nm and emission wavelength 606 nm.

Electron Microscopy SEM images were obtained using a FEI Nova 200NanoLab Dualbeam FIB/SEM (FEI, Hillsboro Oreg., USA), operating at 2-30kV and 10-50 pA.

Sample preparation for TEM Small pieces (˜1-2 mm3) were shaved from alarger biocomposite sample using a #11 scalpel. The pieces were placedin a petri dish and fixed for 1 hour with 2% OsO₄ in dH2O. The pieceswere rinsed 3× with dH2O, dehydrated into acetone over 48 hours, theninfiltrated with Epon-Araldite resin (Electron Microscopy Sciences, PortWashington PA) for 48 hours. Samples were placed in rubber embeddingmolds and the resin polymerized at 60° C. for 24 hours.

Semi-thick (400 nm) serial sections were cut with a UC6 ultramicrotome(Leica Microsystems, Wetzlar, Germany) using a diamond knife (DiatomeUS, Port Washington, PA). Sections were placed on Formvar-coatedcopper-rhodium 1 mm slot grids (Electron Microscopy Sciences) andstained with 3% aqueous uranyl acetate and lead citrate. Colloidal goldbeads (10 nm) were placed on both sides of the section to serve asfiducial markers for subsequent image alignment.

Grids were placed in a dual-axis tomography holder (Model 2040, E.A.Fischione Instruments, Inc., Export, Pa.) and imaged with a TF-30STelectron microscope (ThermoFisher Scientific, Waltham, Mass.) at 300KeV. Images were recorded with US1000 camera (Gatan, Inc.). Montagedprojection images and tomographic tilt-series were acquiredautomatically using the SerialEM software package (41). Briefly, gridswere tilted +/−64° and images acquired at 1° intervals. The grid wasthen rotated 90° and a second tilt series was taken about the orthogonalaxis. Tomographic data were calculated, analyzed and modeled using theIMOD software package (42). 3D reconstructions of selected areas werecreated using the isosurface function of IMOD with the followingparameters: threshold at 133, outer limit 21, X 790, Y 1451, Z 143, Xsize 152, Y size 152, Z size 55, binning 1, smoothing 1, delete smallpieces <100.

Raman Spectroscopy Raman spectra were collected with a Renishaw inViaRaman microprobe (Renishaw, Wotton-under-Edge, United Kingdom) equippedwith a Leica DM 2500 M microscope (Leica Microsystems, Wetzlar,Germany), an Olympus LM Plan FL 20× long working distance objective(numerical aperture=0.4) (Olympus, Tokyo, Japan), a 1200 lines mm-1grating, and a CCD detector configured in a 180° backscatter geometry. A532 nm diode-pumped solid-state (DPSS) laser (Renishaw RL532C50) wasused as the excitation source and a 30 mW radiant flux was incident onthe surface of the sample. A λ/4 plate was used to circularly polarizethe incident excitation. No polarizing collection optics were used.

Spectra of cells suspended in growth media, and reference spectra ofpure water-based growth media were collected. The hydrogen bonding broadpeak at 3300-3400 cm⁻¹ is observed in both cases, but in the case ofsuspended cells the intensity is over 2.5 times higher, indicating thepresence of hydrogen bonding interactions between cellulose fibers.

Thermal Analysis Thermogravimetric analysis measurements were conductedin a D550 TGA from TA Instruments (New Castle, DE). Samples were heatedfrom room temperature to 1000° C. at a heating rate of 1° C./min in N2flow at 40 ml/min.

X-ray Diffraction (XRD) X-Ray diffraction patterns were collected usingPANalytical X'Pert Pro (operating voltage at 40 kV, current at 40 mA,CuKα, λ=0.1541 nm). An angular range of 2θ=10-60° with a step size of0.1° and a scanning speed of 0.008° s⁻¹ was used for the measurements(Panalytical B. V., Holland).

Chemical Analyses Compositional analysis was carried out using theanthrone-sulfuric acid colorimetric method for cellulose, acidichydrolysis for hemicellulose, the carbazole colorimetric method forpectin and the klason method for lignin.

Electrical properties Copper tape was used for the electrodes, connectedto a Keithley 2636B source (Tektronix, Inc. Beaverton, Oreg.). Voltagescans between −2 and 2 V, with a 0.1 V/sec step were recorded. The slopeof the obtained IV curves was converted to conductivity when multipliedwith sample height and divided by cross-sectional area.

Water uptake-methods Water uptake and thickness swelling tests wereperformed according to ASTM D1037 with appropriate modifications (15).Ten (10) dry samples in dimensions of approximately 4×4×1.5 mm wereimmersed in 5 mL of distilled water, and their relative mass andthickness increase were measured after 2 or 24 hours in the distilledwater, after 10 minutes of drying in air.

Mechanical properties 3-point bending and tension tests 3-point bendingand tension tests were performed in an eXpert 8612 axial-torsion tester(Admet Norwood, MA), an Instron 5500 (Norwood, MA) and an Instron E3000(Norwood, Mass.) equipped with 250 N, 500 N, 25 kN and 50 kN load cells.The biocomposite samples were tested with the 250 N (for tensile tests)and 500 N load cells (for bending tests). The larger load cells wereused for reference materials, namely plastics (to accommodate largertensile deformations) and wood samples of larger dimensions.

Mechanical properties-Flexural tests For the flexural tests, a minimumof five samples of each material were tested at a constant strain rateof 0.004±0.001 s−1 until failure. The support span was approximately 30mm for the biocomposite samples. Samples of approximately 40×5×5 mm(length×width×thickness) dimensions were tested in two perpendiculardirections, as shown in FIG. 20 . Natural wood samples were cut in35×7×4 mm strips, and plastic samples were cut at 75×7×4 mm strips. Allreference materials were tested at the same strain rate as thebiocomposites.

Mechanical properties-tensile tests For the tensile tests a minimum offive samples were tested at a constant strain rate of 0.0025±0.0001 s−1until failure. Medium-density fiberboard end-tabs with dimensions10×10×3 mm in all 40×5×5 mm specimens were applied, using a thin layerof polyvinyl acetate adhesive (Gorilla Wood Glue, Cincinnati, Ohio)(43). The natural wood tension samples, with dimensions approximately100×15×5 mm, and the plastic samples in a dog-bone configuration of115×1.5×6 mm (ASTM D638, type IV), were tested at the same strain rateas the biocomposites.

Mechanical properties—compression tests For the compression tests of thebiocomposite samples, approximately 9×9×2 mm (length×width×thickness)samples were tested under compression at a constant rate of 0.001 s−1 toa 10% target strain. The compressive modulus was calculated from thelinear part of the unloading stress-strain curve, while the maximumstress value during compression, corresponding to the 10% strain, wasreferred to as compressive strength.

Example 1: Cell Cultures

Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells were purchasedfrom DSMZ. Cells were kept in Linsmaier & Skoog medium with vitamins(HIMEDIA-PT040) with 3% (w/v) sucrose at a pH of 5.8. The followingsupplements were added: 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1μM a-naphtaleneacetic acid, and 1.46 mM KH2PO4. The cells were grown in50-300 ml aliquots in 100 ml-1 L Flasks on a rotary shaker (130 rpm) atroom temperature and were subcultured biweekly at 1:10-1:60 dilutions.

Alternatively, Tobacco (Nicotiana tabacum) BY-2 (Bright Yellow-2) cells(Nagata et al. 1992) were cultured in Murashige and Skoog (1962) medium,pH 5.8, with the following supplements: 0.9 μM 2,4-dichlorophenoxyaceticacid (2,4-D), 3% (w/v) sucrose, 3 μM thiamine-HCl, and 2.7 mM KH2PO4.The cells were grown in 50 mL aliquots in 300 mL Erlenmeyer flasks on arotary shaker (100 rpm) at 25° C., in the dark, and subcultured weeklyby transferring 1.5 mL of the culture into 50 mL of fresh medium.

Example 2: Structure of Plant Cell Wall

The cell wall in living plant cells extracted from suspension culturesaccording to Example 1 was characterized by optical laser microscopy andby Raman analysis as indicated in the materials and methods. Inparticular, confocal Raman spectroscopy was performed on suspensions ofliving cells to evaluate the chemical composition of the cell walls.

The results are illustrated in FIG. 2 (optical and laser microscopy) andFIG. 3 (confocal Raman spectroscopy).

In particular, the optical and laser microscopy results reported in theillustration of FIG. 2 , show that viable cells are elongated, with amean length of 170±60 μm, a mean width of 45±10 μm, and are surroundedby a thin primary cell wall containing cellulose, pectin and phenoliccompounds (FIG. 2 Panels B-D).

Raman spectroscopy of living cells (FIG. 3 ) reveals the predominantvibrations of cellulose, hemicelluloses, pectin, and the ligninprecursors coniferyl alcohol and coniferaldehyde (23, 24).

In particular the predominant carbohydrate vibrations detected throthrough Raman spectroscopy and shown in FIG. 3 were assigned tocellulose and hemicelluloses including the peaks at 1003 and 1127 cm⁻¹,corresponding to C—C and C—O stretch modes (25, 45, 46). The peaks 1095,1157 and 1338 cm⁻¹ are assigned to C—C and C—O vibrations in cellulose(25, 47). The skeletal vibrations of alpha-anomers of pectin give riseto the peak at 854 cm-1 (23). The 1584 cm-1 peak is attributed to C═Caromatic symmetric stretch vibrations, while the 1657 cm-1 is related toC═C symmetric stretch of coniferyl alcohol and C═O stretch ofconiferaldehyde (23, 48). Therefore, the two latter peaks highlight thepresence of monolignols, which are known lignin precursors, in the plantcell wall (24).

Additionally, in the illustration of FIG. 3 the hydrogen bonding regionis evident from the peak around 3300 cm-1, which is related to thevibrations of both the inter- and intra-chain hydroxyl groups (25). Inorder to bind together neighboring cells, the self-adhesive propertiesof cellulose nanofibers (26) was utilized.

As demonstrated in all-cellulose materials, the native hydrogen bondingalong with van der Waals interactions provide adhesion betweennanocellulose fibrils, thereby eliminating the need for an externaladhesive binder (27).

Example 3: Biocomposite Fabrication

Living cells cultured as described in Example 1 were used to provide abiocomposite according to an exemplary embodiment of the method of theinstant disclosure.

The living cells were harvested from the cell culture. If cells are tobe extracted from a suspension culture, the cultured biomass is a vacuumfiltered (e.g. in an Erlenmeyer flask), to remove water and growth mediafrom the biomass and separate the plant biomass from growth media. Forsolid culture vacuuming is not required. The collected biomass istypically weighed.

If addition of fillers or other additives is desired, such addition isperformed typically after separation of the plant biomass from thecultured biomass. Mixing of the additives with the plant biomass can betypically performed either manual or with assistive equipment (e.g.stirrer, and ultrasonication) to homogenize the mixture.

If control of water content is needed or desired, the plant biomass canbe dried typically following weighing and/or the optional additivesaddition. Oven drying at temperatures below 100° C. for the duration canbe performed to reach the water loss desired. Biomass or hybrid mixtureincluding biomass and additives can typically have the form of solidcell cluster can then be collected and transferred into the mold.

The collected biomass can be placed in particular on porous aluminumcustom designed molds (Metapor Aluminum, Attleboro, Mass.) and subjectedto a controlled compression.

Calibration of the pressure rate dictates the appropriate compressionprotocol. In the case of tobacco cells and their composites a pressurecan be used of 0.1±0.05 MPa/day till 0.8 MPa to compact the dehydratingcells until they reaches a 15±5 wt % solid residual mass (typicallywithin 5-7 days). The samples were held at the maximum pressure (0.8MPa) typically for 2 days until a solid residual mass 10±5 wt % isreached. All samples were subsequently dried for 48 h at 60° C. in abenchtop oven to reach a final dry solid residual mass of 2±1 wt %(Heratherm, Thermo Scientific, Waltham, Mass.).

Material is extracted from the mold after a mass loss of more that 50%is achieved, and dried for a minimum of 12 h at a temperature below 100°C.

The residual water after the fabrication process was 7±3 wt % asdetermined from TGA measurements of the dried samples.

Each sample density was calculated from the ratio of their mass(analytical balance XS205, Mettler Toledo, Columbus, Ohio) divided bytheir volume. In particular, a cultured biomass was extracted from aculture according to Example 1 in a hydrated state (water content 10 wt% or higher).

Example 4: SEM Imaging of Biocomposite Material

The structure of biocomposite materials obtained with the methodexemplified in Example 3 from undifferentiated Nicotiana tobacco BY-2cells was imaged with cross-section scanning electron microscopy (SEM).

The related SEM images are shown in FIG. 4 and FIG. 5 in comparison withSEM images of other material taken at the same magnification.

In particular, FIG. 4 Panel A) illustrates the obtained microstructureof a biocomposite obtained starting from undifferentiated Nicotianatobacco BY-2 cells. It consists of a layered structure of compactedcells. The adjacent cells are in cotact and appear interconnected witheach other, and cannot be distinguished as separate entities by SEMimaging. Also the cell walls cannot be distinguished by SEM.

The structure of the biocomposite material was compared to natural wood(walnut, FIG. 4 Panel B), commercial medium density fiberboard (MDF,FIG. 4 Panel C), and plywood (FIG. 4 Panel D).

From the comparison the biocomposite material obtained appearsstructurally similar to plywood and MFD, which are compressed woodcomposites bound together with polymer adhesives.

However natural wood has a cellular structure with pores and a distinctarchitecture which differentiates it with the pore-less compactedbiocomposites prepared here. MDF and plywood SEM images are more similarto the biocomposites, because they are also compacted structures.However, they do not have the distinct lamellar binder-lessmicrostructure we see in our biocomposites. They rather have aglued-wood fiber structure (FIG. 4C) or a “continuum-phase” of woodplies glued together (FIG. 4D). In our biocomposites the collapsed cellsare not distinguishable but the microstructure is not as homogeneous asin the other two cases where the biomass is glued together.

This is also confirmed by the illustration of Figure FIG. 5 , panel (A)which demonstrate the lamellar and dense microstructure thus obtainedfrom the compacted plant cells which has a material density of 1.1±0.1g/cm³.

Natural wood (for example walnut, FIG. 5 , panel (B)) has a complexcellular microstructure with porosity that varies across the graindirection, while plastics (polypropylene presented in FIG. 5 , panel(C)) have a continuous un-structured appearance. Fibers and/or particlesfrom agricultural waste or alternative biomass can be added beforecompression in order to obtained biocomposite materials.

Example 5: Optical SEM and TEM Images of Biocomposite Material

An exemplary biocomposite obtained according to the procedureexemplified in Example 3, was obtained and the related structureobserved optically, trough SEM imaging and TEM imaging techniquesindicated in the materials and methods. The results are shown in FIG. 6and FIG. 7 .

Optical and SEM observations of the biocomposite panels reveal ananisotropic, dense, lamellar microstructure comprised of compacted plantcells (FIG. 6 Panels A-B). Transmission electron microscopy (TEM)demonstrates that the primary cell walls are preserved during cellcompression and dehydration (FIG. 6 Panels C-D).

Accepted models suggest that the primary cell wall is a multi-lamellatedstructure consisting of cellulose microfibrils, arranged in variousorientations within each plane (from entirely isotropic to somewhataligned, depending on cell type), bound in a matrix of hemicellulosesand pectins (30).

Even in the case of randomly distributed cellulose microfibrils in theplane of the wall, the structure is considered highly anisotropic acrossthickness (30). TEM images of herein described exemplary biocompositesshow an average dehydrated cell wall thickness of 185±57 nm, andcellulose microfibrils diameters ranging between 1 and 30 nm.

High resolution TEM (HRTEM) images confirm the presence ofmulti-lamellated structures, with cellulose microfibrils laying acrossthe consecutive parallel planes (FIG. 6 Panel D, FIG. 7 Panels A-B).Using 3D tomographic reconstructions, the spatial distribution of thecell wall components was analyzed, and their fibrous organization acrossmultiple parallel planes, resulting in a highly anisotropic network(FIG. 7 Panel C) was observed.

A hierarchical microstructure was observed: at the cellular level, alamellar architecture consisting of compacted cells (FIG. 6 Panel B); atthe sub-cellular level, an anisotropic, multi-lamellated structure,derived from the natural organization of the cell wall components (FIG.6 Panels C-D, FIG. 7 Panels B-C).

Example 6: TGA Analysis of Biocomposite Material

A compositional analysis of the dry material obtained according to theprocedures exemplified in Example 3 was performed by thermal gravimetricanalysis (TGA) with the materials and method described. The results areshown in FIG. 8 .

TGA curves of the biocomposite reveal four distinct mass loss steps(FIG. 8 ) as shown in regions I-IV. The first derivative of mass lossTGA curve (DTG) peaks correspond to: evaporation of bound water (peak1), and degradation of pectins (peak 2), hemicelluloses (peak 3),cellulose (peak 4), and phenolic compounds (peak 5-6) (28). The charresidue is 10±5 wt %.

The results shown in FIG. 8 indicate that the dry material is composedof 15% cellulose, 20% hemicelluloses, 6.8% pectins and 6.3% lignols.Thus, the obtained material is a biocomposite, comprised of aheterogeneous mixture of naturally synthesized biopolymers.

Before all testing, all samples were subjected to a 48 hours dehydrationin 60° C. TG analysis reveals that even after the extensive dryingprocess, there is water remaining in AW samples (7±3 wt %), as shown bythe initial mass loss step from 50 to 130° C. (peak 1, region I). Themain mass loss step occurs between 130-500° C. (region II) followed by amonotonous smaller mass loss (region III) and a final mass loss at ahigher rate above 800° C. (region IV). The amorphous pectins andhemicelluloses decomposition is initiated at lower temperatures than thehigher molecular weight components (Mendu et al., 2011; Meng et al.,2015).

In biocomposites AW, the first DTG peak around 180° C. corresponds topectins degradation, while hemicelluloses, cellulose and lignolsdecomposition yield the peaks around 215, 275, and 350 respectively(Gasparovic, Korenova and Jelemensky, 2010; Mendu et al., 2011; Meng etal., 2015). Phenolic compounds have the highest thermal stability anddecompose slower than the rest of the components often at temperaturesbetween 800-900° C., as observed in the AW samples (region IV) (Meng etal., 2015).

Example 7: XRD Analysis of Biocomposite Material

From XRD patterns of biocomposites prepared according to the procedureexemplified in Example 3 were detected. The results are shown in FIG. 9.

The analysis of the diffraction patterns shown in FIG. 9 , reveals thatcellulose exists in multiple crystal polymorphs (I, II and III, markedin FIG. 9 ) (29, 49, 50). The main diffraction peaks from nativecellulose, Iα and Iβ, correspond to (110) and (200) crystallographicplanes respectively, and contribute to the broad 22-23° peak.

The shoulders at 15° and 17° respectively are assigned to Iα and Iβdiffractions from the (100), (010) and (1-10), (110) planes (27).Distinct contributions from cellulose II include the (110) peak at 19.9°and the (1-10) peak at 12.2°. Cellulose III diffractions from the(1-10), (100) and (010) planes give rise to the peaks at 20.9°, 20.6°and 11.6° respectively (29).

Additionally, polysaccharide derivatives of mannose and galactose, whichare backbone constituents of hemicellulose, give rise to the peaks at19.1° and 24.8° (51, 52).

Accordingly, the presence of semicrystalline cellulose is confirmed bythe exemplary XRD of FIG. 9 .

Example 8: Density of Biocomposite Materials \

The density of a biocomposite prepared according to the procedureexemplified in Example 3, was tested for density according to thetechniques indicated in the material and methods above. The relatedresults are shown in FIG. 10 .

In particular the results illustrated in FIG. 12 shows density of thebiocomposite and reference materials. Samples notation: BC: pure(without fillers) bio-composite; 1: pine; 2: poplar; 3: oak; 4: walnut;5: plywood; 6: MDF; 7: PS; 8: PP; 9: LDPE.

Example 9: Biodegradation of Biocomposite Materials

Biodegradability of the biocomposites prepared with a procedureaccording to the indications of the exemplary procedure reported inExample 3, was characterized as indicated in the materials and methodsin comparison with pine natural wood.

In particular, a rectangular shaped pieces of 0.05±0.01 grams of the wasincubated in agricultural soil (FoxFarm Ocean Forest Potting Soil). Eachpot was stored in at outdoors location for 14 weeks. The biocompositesand natural wood control samples were recovered every 2 weeks to measuretheir residual mass. Following literature reported process (44), thesamples were recovered, cleaned and dried at room temperature andsubsequently weighed. The mass loss of the biocomposite was comparedwith that of natural wood (36).

The results are illustrated in FIG. 11 and show an initial mass gaincorresponding to water uptake from the soil, in both natural wood andbiocomposites (FIG. 11 ).

The detectable mass loss due to biodegradation of the biocompositesbegins 3 weeks after incubation, while for natural wood it begins about7 weeks later. This can be associated to the presence of lignin innatural wood, which provides resistance to pathogen attacks on cellwalls (37). An almost complete biodegradation of the biocomposite wasobserved 14 weeks after initial incubation. A potential drawback ofrapid biodegradation is water sensitivity.

Example 10: Mechanical Properties of Biocomposite Materials

Biocomposite materials obtained with the procedures exemplified inExample 3 were tested for mechanical properties in accordance with theprocedures indicated in the material and methods of this section.

Tensile and 3-point bending tests were performed to characterize themechanical performance of the dehydrated biocomposites.

The mechanical properties of different woods and plastics were compared.IN particular, the dehydrated biocomposites were compared to differentsoftwoods (pine), hardwoods (poplar, oak, and walnut), commercialplywood and MDF, and synthetic plastics of similar density (polystyrene,PS, polypropylene, PP, and low-density polyethylene, LDPE) (FIGS. 12-13, FIGS. 14-15 ) and with literature-reported biocomposites from living,eukaryotic organisms (FIG. 16 ).

Tension tests show that herein described exemplary biocomposites arestiffer than the other materials (FIG. 12 ).

However, natural woods have higher strength (FIG. 13 ), which can beexplained by their different cellular architectures, cell wallcompositions, and components arrangements within the secondary cellwalls. The cells used in herein described exemplary biocompositesoriginate from the herbaceous plant Nicotiana tabacum and they naturallydevelop a thin, unlignified primary cell wall (only a low monolignolamount of 6.2 wt % was detected). These cells do not form secondary cellwalls and cannot self-organize in a hierarchical micro-structure inherein described exemplary cultures. Regardless, the mechanicalperformance of herein described exemplary biocomposites is comparable tothat of commercial engineered woods and plastics. They surpass allliterature-reported values for materials composed of plant cells,mycelium, or yeast matrixes (9, 10, 15, 31-33) (FIG. 16 ).

Tension tests of several types of natural woods and plastics show thatthe Young's modulus of AW is 38-64% higher than the tested natural woodsand 47-90% higher than the tested plastics (FIG. 17 Panel B). In termsof tensile strength, AW outperforms PS and LOPE, by 17 and 58%respectively, while PP has an almost double strength compared to AW(FIG. 17 Panel C). Natural woods have considerably higher tensilestrengths compared to AW, with values ranging between 35-110 MPa for thedifferent tested species. Flexural tests show that AW (along thethickness direction) has a 50-95% higher modulus than the testedplastics as disclosed herein. With respect to natural wood (also testedon the higher stiffness direction, along the grain), AW has a 23-38%lower modulus than pine and oak, while poplar and walnut have notablyhigher moduli, in the order of 10 and 20 GPa respectively. The bendingstrength of AW is 30% higher than PS, 76% higher LOPE, and 13% lowerthan PP. The corresponding strength of natural woods is significantlyhigher than AW, with values ranging between 100-160 MPa.

Stress-strain plots obtained from the biocomposites (FIGS. 18-19 ), showan initial linear elastic response upon loading, both under tension andbending, followed by a brittle failure at small strains (1±0.3%).

The Young's modulus, calculated from the initial linear elastic part ofthe tension experiments, is 2.5±0.4 GPa, and the ultimate strength is21.2±3 MPa. The flexural modulus is 4.2±0.4 GPa, and the modulus ofrupture is 49.3±3.2 MPa.

Testing the flexural properties of the biocomposite on the twoperpendicular planes (see schematic in FIG. 20 ), reveals that stiffnessvaries by a factor of ca. 1.75 in the two directions, while strengthremains unaffected by orientation. The measured difference in stiffnessis due to the anisotropic micro-structure of the biocomposite, resultingfrom the fabrication process which orients the cells normal to thecompression direction.

Example 11: Preparation of Biocomposite Including Fillers

Biocomposite materials comprising additives was provided by performing aprocess in accordance with the approach exemplified in Example 3,modified to additives were added.

In particular, a cultured biomass was extracted from a culture accordingto Example 1 in a hydrated state (water content 10 wt % or higher).

The biomass was then mixed with other materials mixtures will be preparein this step. Mixing either manual or with assistive equipment(including stirrer, ultrasonication) are used to homogenize the mixture.

Example 12: Properties Biocomposite Material Including Filler Additives

Mechanical properties of biocomposite materials prepared according tothe methods exemplified in Example 11 were tested with techniquesdescribed in the materials and methods. The related results are reportedin FIG. 21 and FIG. 22 .

In general use the natural biopolymer mixture as a matrix andincorporate filler additives, can be performed to (i) introduce newproperties/functions in the composites, and (ii) enable further tuningof the mechanical performance.

The addition of different amounts of carbon fibers (CF), for example,changes the biocomposites' compressive modulus and strength (FIG. 21 ).For CF concentrations below 5 wt % there is a gradual improvement ofelastic modulus and strength, in the order of 20-25%, followed by adecrease for higher concentrations, as observed in polymer compositesbecause of fillers' aggregation (38).

Similarly, there is a small strength enhancement of about 12% at 5 wt %of carbon fibers, followed by a monotonic decrease at higher fillerloadings. This is a behavior typically observed in polymer compositematerials, in which at low filler concentrations a more efficient fillerdispersion can be obtained, thereby enabling successful load transferbetween the two components. In contrast, at higher concentrations, thefiller particles aggregate, thus contributing to an inhomogeneous stressdistribution upon loading which leads to overall inferior mechanicalperformance (Roumeli et al., 2014).

Different filler particles expand the biocomposites' property space(FIG. 22 ). Elastic modulus as a function of density of differentplant-based biocomposites are plotted in illustrations showing pure cellmatrix (BC), biocomposites containing various amounts of CF, halloysiteand montmorillonite nanoclays (NC) and graphene (G). Their propertieslie at the intersection of natural cellular materials, including “woodproducts”, and commercial plastics (FIG. 22 ), presenting elastic modulispanning over one order of magnitude.

Filler additives also endow new functionalities, such as electricalconductivity or magnetic properties. The electrical conductivity ofplant cell/CF composites, for example, can be tuned varying the CFcontent (FIG. 23 ), in which the IV plots of AW containing 1 and 20 wt %of CF illustrate the effective tuning of electrical conductivity from2.25×10⁻⁷ S/m to 2.2×10⁻³ S/m.

Similarly, the addition of 13.5 wt % iron oxide nanoparticles (IN) inthe plant cell matrix conveys ferro-magnetic properties, which allow thebiocomposite to support more than five times, or six times its weightwhen attracted by a magnet (FIG. 24 ).

Biocomposites have the potential to fulfill increasing global materialdemands from renewable resources and with reduced lifecycleenvironmental impacts (1, 2). Greener biocomposites with higherbiological content, primarily derived from crops and plant fibers, arecontinuously emerging (3, 4). However, most solutions rely onpetrochemicals-based matrixes or binders, or depend on harsh thermal,mechanical and chemical treatment of mature plants (2).

Most recently, self-growing biocomposites have been proposed as a newclass of multi-functional materials, which capitalize on the innateability of living matter to self-fabricate and replicate (5, 6).Bacteria have been used as a fabrication platform for cellulose (6, 7)and polyesters (8) to provide alternative materials to petroleum-basedplastics. More complex living organisms, such as fungi and fermentingyeast have been used in biocomposites (9-11). Remarkably, myceliummaterials already reached the market for protective packaging,insulation, and acoustic panels (9, 12-14). However, the main drawbackof biocomposites that utilize eukaryotic biological growth for biomassproduction is that they have low mechanical performance (9, 15), whichrenders them unsuitable for many engineering and structuralapplications.

Example 13: Water Uptake-Test of Biocomposite Materials

Water uptake and thickness swelling tests were performed onbiocomposites prepared according to the procedures exemplified inExample 3 and Example 11, to dry biocomposite samples (see materials andmethods above).

A water uptake of 191±53% (quantified in weight % gain) were measuredafter 2 hours of immersion. Similarly, the thickness swelling after 2hours is 173±48% (quantified in volume % increase). In this timeframe,the water sensitivity of biocomposites as described herein is similar toother wood-based composite materials (15). The results demonstrate thatthe adhesion between the mechanically compressed cells is poor. Nochemical treatments were used in this experiment to initiate bondingbetween neighboring cells. When used in wet or humid environments, it iscontemplated that surface treatments or coating of the biocompositescould be employed to mitigate water infiltration and damage.

Additional tests on water absorption and thickness swelling wereconducted to dry AW samples, and a water uptake of 260±15% was measuredafter 2 hours of immersion and 415±80% after 24 hours. Similarly, thethickness swelling after 2 h is 225±15%, and after 24 hours it is345±20%. The results demonstrate that the adhesion between themechanically compressed cells can be enhanced by chemical treatment toinitiate any type of bonding between neighboring cells other thanphysical attraction (Sun et al., 2019).

Water uptake and thickness swelling tests indicated that biocompositesas described herein respond similarly to other wood-based materials(15). In applications, the water uptake is mitigated with surfacetreatments or water-resistant coatings.

Experiments performed with biocomposite fabricated as indicated in thisexample with fillers resulted in comparable results.

In summary described herein are biocomposites and related compositions,fabrication methods and systems the biocomposites comprising compactedplants and/or algae cells having a water content of less than 15 wt %,and a minimized pore presence and/or dimensions, in which the compactedcells are in a lamellar stacked configuration with a plurality oflamellae arranged one above the other, each lamella independently havinga thickness of 20 nm to 5 μm and comprising a semi-crystalline structureformed by biopolymers of cell walls of the compacted plant and/or algaecells.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the binding compounds, compositions, devices,methods and systems for the selective detection, and are not intended tolimit the scope of what the Applicants regard as their disclosure.Modifications of the above-described modes for carrying out thedisclosure can be used by persons of skill in the art and are intendedto be within the scope of the following claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles including related supplemental and/orsupporting information sections, abstracts, laboratory manuals, books,or other disclosures) in the Background, Summary, Detailed Description,and Examples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed can be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. The term “plurality” includes two ormore referents unless the content clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible sub-combinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure.

Whenever a range is given in the specification, for example, atemperature range, a frequency range, a time range, or a compositionrange, all intermediate ranges and all subranges, as well as, allindividual values included in the ranges given are intended to beincluded in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably canbe practiced in the absence of any element or elements, limitation orlimitations, which is not specifically disclosed herein.

“Optional” or “optionally” means that the subsequently describedcircumstance can or cannot occur, so that the description includesinstances where the circumstance occurs and instances where it does notaccording to the guidance provided in the present disclosure.Combinations envisioned can be identified in view of the desiredfeatures of the device in view of the present disclosure, and in view ofthe features that result in the formation.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods can include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claim.

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The invention claimed is:
 1. A biocomposite comprising compacted plantand/or algae cells having a water content of less than 15 wt %, thecompacted plant and/or algae cells being poreless or having pores with adiameter of less than 10 μm, the compacted plant and/or algae cellsbeing in a lamellar stacked configuration with a plurality of lamellaearranged one above the other, each lamella independently having athickness of 20 nm to 5 μm, and comprising a semi-crystalline structureformed by cell walls of the compacted plant and/or algae cells, thesemi-crystalline structure having a crystalline component with aglucose-based biopolymer in a matrix of amorphous sugar-basedbiopolymers and/or phenol-based biopolymers of the cell walls.
 2. Thebiocomposite of claim 1, wherein the compacted plant and/or algae cellshave pores with a diameter of less than 5 μm or less than 3 μm.
 3. Thebiocomposite of claim 1, wherein each lamella independently has athickness of 20 nm to 300 nm.
 4. The biocomposite of claim 1, whereineach lamella independently has a thickness of 100 nm to 300 nm.
 5. Thebiocomposite of claim 1 wherein the water content is less than 10 wt %.6. The biocomposite of claim 1, wherein the crystalline component with aglucose based biopolymer comprises cellulose, alginic acid or calciumalginate, a polysaccharide of beta-1,3-xylan, or a polysaccharide ofbeta-1,4-xylan, and/or a polysaccharide of beta-1,4-mannan.
 7. Thebiocomposite of claim 1, wherein the sugar-based biopolymers and/orphenol-based biopolymers comprise hemicellulose, pectin, ligninol,carrageenan, agar, a polysaccharide containing at least one ofxylogalactoarabinan, glucuronoxylorhamnan, rhamnoxylogalactogalacturonanand 3-deoxylxo-2-heptulosaric acid, or any sulfated derivative thereof.8. The biocomposite of claim 1, wherein the plant and/or algae cellscomprise plant cells, the crystalline component with a glucose-basedbiopolymer comprises cellulose fibrils; and the sugar-based biopolymersand/or phenol-based biopolymers comprise hemicellulose, pectin andoptionally ligninol or lignin.
 9. The biocomposite of claim 1, whereinthe plant and/or algae cells comprise algae cells, the crystallinecomponent with a glucose based biopolymer comprises a polysaccharideand/or a glycoprotein; and the sugar-based biopolymers and/orphenol-based biopolymers comprise carrageenan, agar, and/or apolysaccharide containing xylogalactoarabinan or sulfatedxylogalactoarabinan.
 10. The biocomposite of claim 1, wherein the plantand/or algae cells comprise tobacco cells, Arabidopsis cells, and/orChlamydomonas cells.
 11. The biocomposite of claim 1, wherein the plantand/or algae cells consist of plant cells.
 12. The biocomposite of claim1, wherein the plant and/or algae cells consist of algae cells.
 13. Thebiocomposite of claim 1, further comprising up to 40 wt % of anadditive.
 14. The biocomposite of claim 13, wherein the additive is afiller.
 15. The biocomposite of claim 14, wherein the filler is anatural or artificial fiber.
 16. The biocomposite of claim 14, whereinthe filler is an inorganic particulate selected from carbon fiber,graphene, halloysite, montmorillonite nanoclay, iron oxide nanoparticleor a combination thereof.
 17. A biocomposite material fabricationmethod, comprising: compacting along a plane a cultured biomasscomprising cultured plant and/or algae cells from a suspension culture,the cultured plant and/or algae cells having a water content of at least10 wt %, and a turgor pressure, the compacting performed by continuouslyapplying to the cultured biomass an increasing pressure perpendicular tothe plane until reaching an applied pressure corresponding to the turgorpressure of the cultured plant and/or algae cells to provide a compactedbiomass; the method further comprising drying the compacted biomass bya) applying a drying pressure to the compacted biomass, and/or b)applying a drying temperature to the compacted biomass, to obtain thebiocomposite of claim 1 having water content from 0.1 to less than 15 wt%, a mass loss from the cultured biomass of 80-99 wt %, and/or a drydensity of 500-1500 kg/m³.
 18. The biocomposite material fabricationmethod of claim 17, wherein the cultured biomass has a water content ofat least 60 wt %.
 19. The biocomposite material fabrication method ofclaim 17, wherein the increasing pressure comprises a start pressure of5% to 15% of the turgor pressure.
 20. The biocomposite materialfabrication method of claim 17, wherein the increasing pressure is alinearly increasing pressure from 0.1 MPa to 0.8 MPa to 1 MPa.
 21. Thebiocomposite material fabrication method of claim 17, wherein thecontinuously applying to the cultured biomass is performed from 6 to 8days.
 22. The biocomposite material fabrication method of claim 17,wherein the compacted biomass has a water content of at least 50 wt %.23. The biocomposite material fabrication method of claim 17, whereinthe drying pressure is the applied pressure corresponding to the turgorpressure, or a pressure up to 2 or 3 times of the applied pressurecorresponding to the turgor pressure.
 24. The biocomposite materialfabrication method of claim 17, wherein the drying temperature rangesfrom 20° C. to 100° C.
 25. The biocomposite material fabrication methodof claim 17, further comprising mixing at least one additive to thecultured biomass before the compacting.
 26. The biocomposite materialfabrication method of claim 17, further comprising providing thecultured biomass by preparing a suspension culture from cells fromfeedstock biomass comprising agricultural waste and/or agriculturalresidue, algae, dedicated energy crop, forestry residue, waste streamand/or re-useable carbon biosource.
 27. A biocomposite fabricationsystem comprising a cultured biomass comprising cultured plant and/oralgae cells from a suspension culture having a water content of at least10 wt %, and a compression tool, the compression tool configured tointeract with the cultured biomass in accordance with the method ofclaim 17 to provide a biocomposite material having water content from0.1 to less than 15 wt %, a mass loss from the cultured biomass of 80-99wt %, and/or a dry density of 500-1500 kg/m³.
 28. The biocompositefabrication system of claim 27, wherein the cultured biomass comprisescells from a feedstock biomass selected from agricultural residues,algae, dedicated energy crop, forestry residue, waste stream and/orre-useable carbon bio source.
 29. The biocomposite fabrication system ofclaim 28, wherein the compression mold is a porous compression mold. 30.The biocomposite fabrication system of claim 27, wherein the compressiontool is a compression mold.